Tacrine-(hydroxybenzoyl-pyridone) hybrids as potential multifunctional anti-Alzheimer's agents: AChE inhibition, antioxidant activity and metal chelating capacity

Article abstract of DOI:10.1016/j.jinorgbio.2016.05.005

Three novel potentially site-activated multitarget tacrine-(hydroxybenzoyl-pyridone) (TAC-HBP) hybrids were designed, synthesized and evaluated as acetylcholinesterase (AChE) inhibitors, antioxidants and biometal chelators. All of them are dual-binding site AChE inhibitors with activity in sub-micromolar range (IC₅₀?=?0.57–0.78?μM), which is comparable to the parent tacrine, and have good 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (EC₅₀?=?204–249?μM) conferred by the hydroxybenzoyl-pyridone (HBP) moiety. Their chelating capacity towards redox-active and/or amyloid-β-binding metal ions (Fe(III), Cu(II)), Zn(II)) was evaluated by using 2′-hydroxy-4′-methoxybenzoyl-2-pyridone derivative as a model compound in 30% w/w DMSO/water medium. It was proved that the HBP moiety acts as a moderate/good chelator of these biometals (pFe?=?13.9, pCu?=?6.0 and pZn?=?6.0 at pH?6.0, C_L/C_M?=?10, C_M?=?10^??6?M), being able to form complexes with β-phenol-keto coordination mode, and that this chelating ability is preserved in the TAC-HBP hybrids.

Full text of DOI:10.1016/j.jinorgbio.2016.05.005

Tacrine-(hydroxybenzoyl-pyridone) hybrids as potential multifunctional
anti-Alzheimer¡!–[INS]–¿’¡!–[/INS]–¿s agents: AChE inhibition, antioxidant
activity and metal chelating capacity
Karam Chand, Hesham M. Alsoghier, S´ılvia Chaves, M. Ame´lia San-
tos
PII:
DOI:
Reference:
S0162-0134(16)30129-5
doi: 10.1016/j.jinorgbio.2016.05.005
JIB 9994
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
15 December 2015
2 May 2016
10 May 2016
Please cite this article as: Karam Chand, Hesham M. Alsoghier, S´ılvia Chaves,
M. Am´elia Santos, Tacrine-(hydroxybenzoyl-pyridone) hybrids as potential multifunc-
tional anti-Alzheimer¡!–[INS]–¿’¡!–[/INS]–¿s agents: AChE inhibition, antioxidant ac-
tivity and metal chelating capacity, Journal of Inorganic Biochemistry (2016), doi:
10.1016/j.jinorgbio.2016.05.005
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

ACCEPTED MANUSCRIPT
Tacrine-(hydroxybenzoyl-pyridone)
hybrids
as
potential
multifunctional anti-Alzheimer´s agents: AChE inhibition, antioxidant
activity and metal chelating capacity
Karam Chanda), Hesham M. Alsoghiera,b), Sílvia Chavesa)*, M. Amélia Santos a)*
a) Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa,
Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal.
b) South Valley University, Faculty of Science, Chemistry Department, 83523 Qena,
Egypt.
Keywords: Hydroxybenzoyl-pyridones; Tacrine; Anti-neurodegeneratives; Anti-AChE
activity; Anti-oxidant activity; Metal chelation.
* Corresponding authors:
(Sílvia Chaves) E-mail: silvia.chaves@tecnico.ulisboa.pt; Phone: +351-218419269
(M. Amélia Santos) E-mail: masantos@ist.utl.pt; Phone: +351-218419273
Abstract
Three novel potentially site-activated multitarget tacrine-(hydroxybenzoyl-pyridone)
(TAC-HBP) hybrids were designed, synthesized and evaluated as acetylcholinesterase
(AChE) inhibitors, antioxidants and biometal chelators. All of them are dual-binding
site AChE inhibitors with activity in sub-micromolar range (IC50 = 0.57-0.78 M),
which is comparable to the parent tacrine, and have good 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging capacity (EC50 = 204-249 M) conferred by the
hydroxybenzoyl-pyridone (HBP) moiety. Their chelating capacity towards redox-active
and/or amyloid-β-binding metal ions (Fe(III), Cu(II)), Zn(II)) was evaluated by using 2′-
hydroxy-4′-methoxybenzoyl-2-pyridone derivative as a model compound in 30% w/w
DMSO/water medium. It was proved that the HBP moiety acts as a moderate/good
chelator of these biometals (pFe = 13.9, pCu = 6.0 and pZn = 6.0 at pH 6.0, CL/CM = 10,
CM = 10-6 M), being able to form complexes with β-phenol-keto coordination mode, and
that this chelating ability is preserved in the TAC-HBP hybrids.
1

ACCEPTED MANUSCRIPT
1. Introduction
Alzheimer’s disease (AD) is a progressive neurological disease leading to memory
impairment and loss of language, judgment and orientation, eventually causing
incapacitation and death [1]. This is a multifaceted disease for which several factors,
such as cholinergic dysfunction, deposits of amyloid-β (Aβ) and tau (τ)-protein as well
as reduced blood supply in brain, are supposed to be relevant for its development [2].
The brains of AD patients display also other characteristic pathological features, such as
metal ion (Fe, Cu, Zn) dyshomeostasis and elevated oxidative stress [3]. Metal ions,
such as Cu and Zn, bind to Aβ peptides, thereby contributing to enable their
aggregation, while deregulated redox active metal ions, Cu(I/II) and Fe(II/III), stimulate
the overproduction of reactive oxygen species (ROS) with concomitant degradation of
several cellular biomolecules (e.g. proteins, deoxyribonucleic acid (DNA), lipids).
The most frequently prescribed anti-AD drugs are cholinesterase inhibitors (ChEIs) that
act by increasing acetylcholine (ACh) level in the brain via inhibition of
acetylcholinesterase (AChE) and/or butyrylcholinesterase (BuChE) [4]. Tacrine (see
Fig. 1), which was the first drug approved by the U.S. FDA for the palliative treatment
of AD, showed reduction in the decline of cognitive performance due to inhibition of
AChE [5], but it was discontinued because it turned out to be hepatotoxic. Regrettably,
ChEIs have been proving to be inefficient in AD therapy being unable to delay or
prevent the progression of this disease. In fact, due to the complex pathogenesis of AD
and to the possible interconnection of several intervenient factors, an innovative
therapeutic approach based on multi-target-directed ligands (MTDLs) has been applied
in the search for potential ChEIs anti-AD drugs [6]. The most common strategy to
design MTDLs is to connect two distinct classes of compounds in one molecule by
choosing a proper spacer.
Since oxidative stress, as well as metal ion dyshomeostasis, are known to play important
roles in the processes of AD pathogenesis, by preceding (biomolecule disruption) or
provoking hallmark pathologies of this disease (neurofibrillary tangles, senile plaques,
ROS) [7,8] it seems that compounds presenting both radical scavenging activity and
capacity for biometal modulation in the brain may be useful for either the prevention or
the therapy of AD.
2

ACCEPTED MANUSCRIPT
Pyridin-2(1H)-ones (2-pyridones) have long been considered important building blocks
in drug discovery due to the number of biologically active molecules containing this
moiety. Along the last decades, several natural compounds with this core structure have
emerged with numerous pharmacological activities, namely anti-AD (Huperzine A
[9,10], AChEI and antioxidant) and antitumour (e.g. Camptothecin [11]), while many
synthetic or semi-synthetic clinical drugs also enclose that non-toxic moiety [12] (see
Fig. 1).
Therefore, as part of our interest on new polyfunctional drugs to combat AD, by
conjugating an active AChEI moiety (e.g. tacrine) with a variety of functional groups
[13-15], and also looking into the potential clinical application of tacrine and 2-pyridone
moieties, we herein hypothesized the inclusion of a 2-hydroxybenzoyl-2-pyridone
(HBP) derivative on such extra-functionalization. These tacrine-(hydroxybenzoyl-
pyridone) (TAC-HBP) conjugates are expected to: a) exhibit AChE inhibition (tacrine
moiety) and b) be able to tackle other pathways such as the oxidative stress found in the
brain of AD patients, due to excess of redox-active (Cu, Fe) metal ions/free radicals, by
chelators (HBP moiety) able to modulate misplaced redox-active metal ions and
possessing anti-oxidant properties to scavenge free radicals. Under the followed design
strategy, molecular modelling studies were performed of these TAC-HBP hybrids in
which tacrine is linked through different alkyl spacers to the envisioned 2-pyridone
moieties. These 2-pyridone moieties are based on a 5-(2-hydroxybenzoyl)-pyridin-
2(1H)one scaffold with different ring substitutes (compounds I, see Fig. 1) and have
already proved activity against c-Src kinase, over-expressed in various human cancers
[16]. A synthetic approach for the preparation of tacrine conjugates II (see Fig. 1) with
appropriate size linkers, previously established by docking studies, was implemented
and afterwards compounds II were assayed in terms of AChE inhibition and anti-
oxidant capacity - 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
assays - while metal (Fe, Cu, Zn) chelating power was evaluated for a model compound
(I, R=4’-OMe) and confirmed for a TAC-HBP hybrid (II, R=5´-OMe, n=1).
Fig. 1 - Molecular structure of: anti-AD drugs - tacrine and huperzine A –, antitumour
camptothecin and HBP derivatives under study (I, II).
2. Experimental Section
3

ACCEPTED MANUSCRIPT
2.1 Materials and methods
Analytical grade reagents were purchased from current suppliers and were used as
delivered. Whenever necessary, solvents were dried according to standard methods [17].
The aqueous iron (FeCl3, 0.0177 M), copper (CuCl2, 0.015 M) and zinc (ZnCl2, 0.0156
M) stock solutions were prepared from 1000 ppm standards (Titrisol) and their metal
content was evaluated by atomic absorption. The iron stock solution was prepared in
acid chloride excess, to prevent hydrolysis, and its exact concentration in HCl was
accurately determined by the usual standard-addition method using 0.1 M HCl
(Titrisol). This 0.1 M HCl solution, also used in calibration of the glass electrode, was
prepared from a Titrisol ampoule. The titrant used in pH-potentiometric and
spectrophotometric titrations was prepared from carbonate free commercial concentrate
(Titrisol, KOH 0.1 M ampoules). The KOH solution was standardized by titration with
a solution of potassium hydrogen phthalate and was discarded whenever the percentage
of carbonate, determined by Gran’s method [18], was greater than 0.5% of the total
amount of base.
Reactions were monitored by precoated thin layer chromatography (TLC) plates (Merck
silica gel 60F254); the spots were visualized either by UV light, or by spraying with 5%
alcoholic FeCl3 or ninhydrin solutions. Silica gel60 (0.061–0.2 mm) was used for
column chromatography. Melting points were measured with a Leica Galen III hot stage
1
13
apparatus and are uncorrected. The H and C NMR spectra were recorded on Bruker
AVANCE III-400 (400 MHz, 100.5 MHz) and Bruker AVANCE III (300 MHz, 75.5
MHz) NMR spectrometers, at 25 °C using residual peaks of solvents as an internal
standard. The chemical shift values are on a δ scale and the coupling constant values (J)
are in Hertz. The following abbreviations are used: s = singlet, d = doublet, t = triplet, m
= multiplet, dd = double doublet and brs = broad singlet. Peak attribution was,
whenever necessary, confirmed by two-dimensional NMR experiments, namely
correlation spectroscopy (COSY) and heteronuclear single quantum coherence
spectroscopy (HSQC). The antioxidant and AChE inhibitory activities were determined
by using a Perkin Elmer Lambda 35 spectrophotometer equipped with a temperature
°
programmer PTP 1+1 Peltier System, using thermostated (T = 25.00.1 C) 1 cm path
length cells. Electrospray ionization-mass spectrometry (ESI-MS) experiments were
carried out on a LCQ Fleet mass spectrometer operated in the ESI positive and negative
ion modes (Thermo Scientific).
4

ACCEPTED MANUSCRIPT
2.2 Molecular modeling studies of TAC-HBP hybrids
The docking calculations were performed following a procedure identical to that
previously reported [19]. The X-ray crystallographic structure of acetylcholinesterase
Torpedo californica AChE (TcAChE) complexed with an inhibitor was taken from
RCSB Protein Data Bank (PDB entry 1ODC), in order to be used as receptor in the
docking simulations. The original complex structure was treated using Maestro v. 9.3
[20], by removing the original ligand, solvent and co-crystalization molecules and
adding hydrogen atoms. This program was also used to design the ligand structures,
which were submitted to random conformational search (RCS) of 1000 cycles and 2500
optimization steps with the program Ghemical v 2.0 [21]. The ligands were docked into
the AChE structure, using GOLD v. 5.1. [22] with the default parameters of GOLD
(except ‘allow early termination’ option) and the Astex Statistical Potential (ASP)
scoring function. The zone of interest was defined as the residues within 10 Å from the
original position of the ligand in the crystal structure.
2.3 Synthesis of the compounds
2.3.1. General procedure for the synthesis of N-substituted HBP derivatives (6-
11):
To a solution of (4-oxo-4H-chromen-3-yl)acrylate (3-5) (4 mmol) and aminoalkane or t-
butylaminoethylcarbamate (4.1 mmol) in ethanol (50 mL) was added triethylamine (2
drops), and the reaction mixture was refluxed for 8-10 h. The progress of reaction was
monitored on TLC. On completion of reaction, the mixture was cooled to room
temperature, and the solvent was evaporated under reduced pressure. The crude product
was either purified by column chromatography over silica gel (100–200 mesh) in 2-3%
dichloromethane/methanol or through crystallisation in CH3OH-CH3CN system, to give
HBP derivatives (6-11) in 74-85% yield. Although spectral data for compounds 6-8 has
been recently reported [16,23], their full characterization is herein presented, along with
that for compounds 9-11, since the antioxidant activity of compounds 6-8 is evaluated
1
13
on this article. See also Supplementary Information with H and C NMR spectra for
compounds 6-17.
5-(2-Hydroxy-4-methoxybenzoyl)-1-isopropylpyridin-2(1H)-one
(6):
The
title
compound 6 was obtained as a light yellow coloured solid with 85 % yield by following
the literature reported reaction of (E)-ethyl 3-(7-methoxy-4-oxo-4H-chromen-3-
5

ACCEPTED MANUSCRIPT
yl)acrylate (2 mmol) with isopropyl amine (2.2 mmol) [23]; mp = 139-141 oC; 1H NMR
(400 MHz, CDCl3): δ = 1.42 (d, 6H, J = 4.0 Hz, CH(CH3)2), 3.87 (s, 3H, OCH3), 5.26-
5.32 (m, 1H, (CH3)2CH), 6.47 (d, 1H, J = 8.0 Hz, H-5'), 6.52 (s, 1H, H-3'), 6.64 (d, 1H,
J = 8.0 Hz, H-6'), 7.47 (d, 1H, J = 8.0 Hz, H-3), 7.70 (dd, 1H, J = 4.0 & 8.0 Hz, H-4),
7.94 (s, 1H, H-6), 12.24 (brs, 1H, OH); 13C NMR (100.5 MHz, CDCl3): δ = 21.97,
47.52, 55.69, 101.50, 107.59, 112.58, 118.04, 119.47, 133.28, 138.40, 161.78, 165.83,
166.18, 194.01; ESI-MS (m/z): Calculated for C16H17NO4 287.1158, found [M+H]+
288.43 and [2M]+ 574.98; Analysis calc. for C16H17NO4.0.030 CH3CN: C 66.89, H 5.96,
N 4.88, %; found: C 67.16, H 6.04, N 4.91 %.
5-(2,4-dihydroxybenzoyl)-1-isopropylpyridin-2(1H)-one (7): The title compound 7 was
obtained as a light yellow coloured solid with 78% yield by following the literature
reported reaction of (E)-ethyl 3-(7-hydroxy-4-oxo-4H-chromen-3-yl)acrylate (2 mmol)
o
with isopropyl amine (2.1 mmol) [23]; m.p. = 198-200 C; 1H NMR (400 MHz,
DMSO-d6): δ = 1.32 (d, 6H, J = 8.0 Hz, CH(CH3)2), 4.99-5.06 (m, 1H, (CH3)2CH),
6.37-6.40 (m, 2H, H-5' & H-3'), 6.46 (d, 1H, J = 8.0 Hz, H-3), 7.40 (d, 1H, J = 8.0 Hz,
H-6'), 7.69 (d, 1H, J = 8.0 Hz, H-4), 8.11 (d, 1H, J = 4.0 Hz, H-6), 10.48 (brs, 1H, OH),
11.44 (brs, 1H, OH); 13C NMR (100.5 MHz, DMSO-d6): δ = 21.54, 47.80, 103.22,
108.44, 114.21, 117.56, 118.89, 134.19, 138.86, 140.54, 161.37, 162.48, 164.19,
192.83; Calculated for C15H15NO4 273.1001, found [M+H]+ 274.27 and [M-H]+ 272.87.
Analysis calc. for C15H15NO4.0.020 CH3CN: C 65.91, H 5.53, N 5.13 %; found: C
65.99, H 5.58, N 5.12%.
5-(2,5-Dihydroxybenzoyl)-1-isopropylpyridin-2(1H)-one (8): The title compound 8 was
obtained as a light yellow coloured solid with 68% yield by following the literature
reported reaction of (E)-ethyl 3-(6-hydroxy-4-oxo-4H-chromen-3-yl)acrylate (2 mmol)
o
1
with isopropyl amine (2.1 mmol) [16]; mp = 156-158 C; H NMR (400 MHz, MeOD-
d4): δ = 1.40 (d, 6H, J = 4.0 Hz, CH(CH3)2), 5.13-5.20 (m, 1H, (CH3)2CH), 6.57 (d, 1H,
J = 8.0 Hz, H-3), 6.83 (s, 1H, H-6'), 6.86 (d, 1H, J = 8.0 Hz, H-3'), 6.95 (dd, 1H, , J =
4.0 & 8.0 Hz, H-4'), 7.89 (dd, 1H, J = 4.0 & 8.0 Hz, H-4), 8.18 (d, 1H, J = 4.0 Hz, H-6);
13C NMR (100.5 MHz, CDCl3): δ = 21.84, 48.76, 115.94, 118.10, 118.21, 119.36,
119.74, 125.54, 138.69, 139.77, 149.17, 156.14, 162.23, 194.79; Calculated for
C15H15NO4 273.1001, found [M+H]+ 274.26 and [2M]+ 546.71; Analysis calc. for
C15H15NO4.0.010 CH3CN: C 65.91, H 5.53, N 5.13 %; found: C 65.80, H 5.51, N
5.09%.
6

ACCEPTED MANUSCRIPT
t-Butyl 2-(5-(2-hydroxy-4-methoxybenzoyl)-2-oxopyridin-1(2H)-yl)ethylcarbamate (9):
The title compound (9) was obtained from the reaction of (E)-ethyl-3-(7-methoxy-4-
oxo-4H-chromen-3-yl)acrylate (4) with t-butyl 2-aminoethylcarbamate as a light yellow
o
1
solid in 83% yield by following the general procedure. mp: 167-168 C. H NMR (400
MHz, DMSO-d6): δ 1.24 (s, 9H, -OC(CH3)3), 3.24 (brs, 2H, H-2"), 3.81 (s, 3H, OCH3),
3.97 (brs, 2H, H-1''), 6.46 (d, 1H, J = 8.0 Hz, H-5'), 6.52-6.54 (m, 2H, H-3' & H-6′),
6.92 (t, 1H, J = 8.0 Hz, CONH), 7.54 (d, 1H, J = 8.0 Hz, H-3), 7.73-7.76 (m, 1H, H-4),
8.01 (s, 1H, H-6), 11.36 (brs, 1H, OH); 13C NMR (100.5 MHz, DMSO-d6): δ 28.45,
38.70, 50.05, 56.03, 78.36, 101.84, 107.01, 115.44, 116.63, 119.17, 133.62, 139.43,
145.94, 156.13, 161.87, 162.02, 164.87, 192.65; ESI-MS (m/z): Calculated for
C20H24N2O6 388.16, found [M]+ 388.94 and [M-H]+ 387.31.
t-Butyl
2-(5-(2-hydroxy-5-methoxybenzoyl)-2-oxopyridin-1(2H)-yl)ethylcarbamate
(10): The title compound (10) was obtained from the reaction of (E)-ethyl-3-(6-
methoxy-4-oxo-4H-chromen-3-yl)acrylate (5) with t-butyl 2-aminoethylcarbamate as a
light yellow solid in 81% yield by following the general procedure. mp: 145-146 oC. 1H
NMR (400 MHz, DMSO-d6): δ 1.25 (s, 9H, -OC(CH3)3), 3.21-3.23 (m, 2H, H-2"), 3.71
(s, 3H, OCH3), 3.93-3.94 (m, 2H, H-1''), 6.43-6.46 (d, 1H, H-3), 6.82 (s, H-6'), 6.88-
6.90 (m, 2H, CONH & H-3') 7.00 (d, 1H, J = 8.0 Hz, H-4'), 7.73-7.76 (m, 1H, H-4),
8.00 (s, 1H, H-6), 9.66 (brs, 1H, OH); 13C NMR (100.5 MHz, DMSO-d6): δ 28.46,
50.19, 55.96, 56.49, 78.33, 113.97, 116.91, 118.18, 118.98, 119.40, 125.54, 139.12,
146.66, 149.91, 152.42, 156.09, 161.98, 191.70. ESI-MS (m/z): Calculated for
C20H24N2O6 388.16, found [M+Na]+ 411.15 and [M-H]+ 387.05.
t-Butyl (4-(5-(2,4-dihydroxybenzoyl)-2-oxopyridin-1(2H)-yl)butyl)carbamate (11): The
title compound (11) obtained from the reaction of (E)-ethyl 3-(7-hydroxy-4-oxo-4H-
chromen-3-yl)acrylate (3) with t-butyl 2-aminoethylcarbamate as a light yellow solid in
o
1
81% yield by following the general procedure. mp: 190-192 C; H NMR (400 MHz,
DMSO-d6): δ 1.34-1.38 (m, 11H, -OC(CH3)3 & H-3"), 1.59-1.62 (m, 2H, H-2"), 2.91-
2.93 (m, 2H, H-4"), 3.95 (t, 2H, J = 8.9 Hz, H-1''), 6.35 (s, 1H, H-3'), 6.38 (d, 1H, J =
8.0 H-5'), 6.45 (d, 1H, J = 8.0 Hz, H-3), 6.81 (t, 1H, J = 8.0 Hz, CONH), 7.40-7.43 (m,
1H, H-6'), 7.71 (d, 1H, J = 8.0 Hz, H-4), 8.22 (s, 1H, H-6), 10.47 (brs, 1H, OH), 11.41
13
(brs, 1H, OH); C NMR (100 MHz, DMSO-d6): δ 19.00, 26.53, 27.03, 28.69, 49.33,
56.49, 77.91, 103.22, 108.39, 114.15, 117.27, 119.03, 134.25, 139.59, 144.86, 156.10,
7

ACCEPTED MANUSCRIPT
161.73, 162.54, 164.17, 192.89. ESI-MS (m/z): Calculated for C21H26N2O6 402.18,
found [M+Na]+ 425.10 and [M-H]+ 401.10
2.3.2. General procedure for the synthesis of free amino alkyl N-substituted HBP
derivatives (12-14):
To the ice cold solution of the Boc protected HBP derivatives (9-11) (0.5 mmol) in 20
mL dichloromethyl (DCM) was added trifluoroacetic acid (TFA, 0.5 mL) dropwise, and
o
the reaction mixture was stirred at 0-5 C for 30 min. Then reaction temperature was
allowed to attain room temperature and stirred at room temperature (RT) for 7-8 hrs.
The progress of reaction was monitored on TLC. On completion, reaction mixture was
neutralized with 15 mL of 30% ammonia solution. The reaction mixture was then
subjected to extraction by adding 2 g of NaCl (to make the aqueous layer saturated) and
additional 3×15 mL of DCM. The evaporation of combined organic layer under reduced
pressure after drying over anhydrous Na2SO4, resulted into free amino alkyl substituted
HBP derivatives (12-14) in 88-92% yield.
1-(2-aminoethyl)-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one (12): The title
compound (12) was obtained from the hydrolysis of Boc group of compound 9 with
TFA as a greenish yellow solid in 92% yield by following the general procedure. mp:
1
150-152oC. H NMR (400 MHz, DMSO-d6):  2.41-243 (m, 2H, H-2"), 3.83 (s, 3H,
OCH3), 4.05 (brs, 2H, H-1"), 6.46-649 (m, 1H, H-5'), 6.55-6.57 (m, 2H, H-3' & H-6′),
7.54 (d, 1H, J = 8.0 Hz, H-3), 7.74 (d, 1H, J = 8.0 Hz, H-4), 8.19 (s, 1H, H-6), 11.43
(brs, 1H, OH); 13C NMR (100.5 MHz, DMSO-d6): 46.82, 56.04, 57.62, 101.71, 107.02,
116.52, 118.91, 133.61, 139.59, 145.98, 161.92, 164.99, 192.94; ESI-MS (m/z):
C15H16N2O4 288.11, found [M + H]+ 289.23.
1-(2-Aminoethyl)-5-(2-hydroxy-5-methoxybenzoyl)pyridin-2(1H)-one (13): The title
compound (13) was obtained from the hydrolysis of Boc group of compound 10 with
TFA as a greenish yellow solid in 88% yield by following the general procedure. mp:
o
1
159-161 C. H NMR (400 MHz, DMSO-d6):  2.43 (t, 2H, J = 8.0 Hz, H-2"), 3.65 (s,
3H, OCH3), 3.98 (t, 2H, J = 8.0 Hz, H-1''), 6.37-6.40 (m, 1H, H-3), 6.80 (d, 1H, J = 4.0
Hz, H-3'), 6.83-6.85 (m, 1H, H-6'), 6.95 (dd, 1H, J = 4.0 & 8.0 Hz, H-4'), 7.69 (dd, 1H,
13
J = 4.0 & 8.0 Hz, H-4), 8.13 (d, 1H, J = 4.0 Hz, H-6), 9.82 (brs, 1H, OH); C NMR
(100.5 MHz, DMSO-d6): δ 46.80, 55.99, 57.71, 114.33, 116.70, 118.01, 118.72, 119.21,
8

ACCEPTED MANUSCRIPT
125.45, 139.07, 146.94, 150.10, 152.32, 161.83, 191.94; ESI-MS (m/z): Calculated for
C15H16N2O4 288.11, found [M+H]+ 289.15.
1-(4-Aminobutyl)-5-(2,4-dihydroxybenzoyl)pyridin-2(1H)-one
(14):
The
title
compound (14) was obtained from the hydrolysis of the Boc group of compound 11
with TFA as a light yellow solid in 88% yield by following the general procedure. mp
185-187 oC. 1H NMR (400 MHz, DMSO-d6): δ 1.43-1.47 (m, 2H, H-3"), 1.67-1.71 (m,
2H, H-2"), 2.69 (t, 2H, J = 8.0 Hz, H-4"), 3.98 (t, 2H, J = 8.0 Hz, H-1''), 6.11 (s, 1H, H-
3'), 6.19 (d, 1H, J = 8.0 Hz, H-3), 6.44-6.47 (m, 1H, H-5'), 7.30-733 (m, 1H, H-6'),
7.69-7.72 (m, 1H, H-4), 8.16 (s, 1H, H-6), 10.42 (brs, 1H, OH), 11.42 (brs, 1H, OH);
13C NMR (100 MHz, DMSO-d6): δ 26.25, 26.70, 49.02, 56.49, 103.66, 110.81, 111.16,
117.70, 118.98, 134.11, 139.81, 143.87, 161.74, 164.56, 170.44, 191.49. ESI-MS (m/z):
Calculated for C16H18N2O4 = 303.13, found [M+H]+ 303.24.
2.3.3. General procedure for the synthesis of TAC-HBP hybrids (15-17):
Firstly, the mixture of 9-chloro-1,2,3,4-tetrahydroacridine (2, 0.4 mmol) and phenol (0.2
o
mmol) was heated with potassium iodide at 50 C for 10 min. When reaction mixture
became a dense oily material, free aminoalkyl substituted HBP derivatives (12-14, 0.4
mmol) were added to it and reaction mixture was heated at 165-170 oC for next 15 min.
Accomplishment of reaction was monitored on TLC. On completion, the crude reaction
mixture was cooled to RT and purified by column chromatography over silica gel 60
(0.061–0.2 mm) in 4-5% methanol/dichloromethane to give TAC-HBP hybrids (15-17)
in 28-45% yield.
5-(2-hydroxy-4-methoxybenzoyl)-1-(2-((1,2,3,4-tetrahydroacridin-9-
yl)amino)ethyl)pyridine-2(1H)-one (15): Title compound (15) was obtained by reaction
of 2 with amine (12) as a light yellow solid in 28% yield by following the general
o
1
procedure. mp: 259-260 C; H NMR (300 MHz, DMSO-d6): δ 1.80 (brs, 4H, H-2′′′ &
H-3′′′), 2.59 (brs, 2H, H-1′′′), 2.96 (brs, 2H, H-4′′′), 3.82 (s, 3H, OCH3), 4.25-4.26 (m,
2H, H-1′′), 4.35-4.36 (m, 2H, H-2′′), 6.43-6.46 (m, 2H, H-3′ and H-3), 6.52 (d, 1H, J =
3.0 Hz, H-5′), 7.39 (m, 1H, H-4), 7.54 (t, 1H, J = 8.0 Hz, H-7′′′), 7.68 (m, 1H, J = 3.0 &
9.0 Hz, H-6′), 7.77-7.79 (m, 2H, NH & H-5′′′), 7.85 (t, 1H, J = 6.0, H-6′′′) 8.09 (brs, H-
6), 8.35 (d, 1H, J = 9.0 Hz, H-8′′′), 11.21 (brs, 1H, OH); 13C NMR (75.4 MHz, DMSO-
d6): δ 20.72, 21.83, 24.50, 28.51, 46.73, 50.24, 56.06, 101.82, 106.91, 112.38, 115.59,
9

ACCEPTED MANUSCRIPT
116.16, 117.53, 118.95, 119.78, 125.23, 125.75, 133.41, 138.16, 140.08, 145.39,
151.63, 156.43, 161.63, 162.43, 164.82, 192.25; ESI-MS (m/z): Calculated for
C28H27N3O4 [M+H]+ 470.21, found 470.38. Analysis calc. For (C28H27N3O4. 0.025
H2O): C 71.62, H 5.80, N 8.95 %; found: C 71.77, H 5.87, N 8.98%.
5-(2-hydroxy-5-methoxybenzoyl)-1-(2-((1,2,3,4-tetrahydroacridin-9-
yl)amino)ethyl)pyridin-2(1H)-one (16): Title compound (16) was obtained by reaction
of 2 with amine (13) as a light yellow solid in 45% yield by following the general
o
1
procedure. mp: 143-145 C; H NMR (400 MHz, DMSO-d6): δ 1.78 (brs, 4H, H-2′′′ &
H-3′′′), 2.57 (brs, 2H, H-1′′′), 2.94 (brs, 2H, H-4′′′), 3.67 (s, 3H, OCH3), 4.07-4.08 (m,
2H, H-1′′), 4.30-4.32 (m, 2H, H-2′′), 6.41-6.44 (m, 1H, H-3), 6.77 (d, 1H, J = 4.0 Hz, H-
3′), 6.91(d, 1H, J = 8.0 Hz, H-4′), 6.99-7.00 (dd, 1H, J = 4.0 & 8.0 Hz, H-4), 7.27 (brs,
1H, NH), 7.44 (t, 1H, J = 8.0 Hz, H-7′′′), 7.70 (dd, 1H, J = 4.0 & 8.0 Hz, H-4′), 7.74 (t,
1H, J = 8.0 Hz, H-6′′′), 7.83-785 (m, 2H, H-6′ & H-5′′′), 8.15 (d, J = 4.0 Hz, H-6), 8.24
(d, 1H, J = 8.0 Hz, H-8′′′), 9.73 (brs, 1H, OH); 13C NMR (100 MHz, DMSO-d6): δ
21.22, 22.13, 24.65, 29.84, 46.81, 50.38, 55.35, 113.56, 114.01, 117.25, 117.50, 118.21,
118.77, 119.45, 122.08, 123.97, 124.69, 125.18, 125.37, 131.83, 139.76, 146.28, 149.96,
152.36, 153.33, 154.67, 162.48, 191.60; ESI-MS (m/z): Calculated for C28H27N3O4
469.20, found [M+H]+ 470.39. Analysis calc. for (C28H27N3O4. 0.005 H2O): C 71.62, H
5.80, N 8.95 %; found: C 71.66, H 5.93, N 9.03%.
5-(2,4-Dihydroxy-benzoyl)-1-[4-(1,2,3,4-tetrahydro-acridin-9-ylamino)-butyl]-1H-
pyridin-2-one (17): Title compound (17) was obtained by reaction of 2 with amine (14)
as a light yellow solid in 33% yield by following the general procedure. mp: 264-266
oC; 1H NMR (400 MHz, DMSO-d6): δ 1.73 (brs, 4H, H-2" & H-3"), 1.83 (brs, 4H, H-2′′′
& H-3′′′), 2.63 (brs, 2H, H-1′′′), 2.97 (brs, 2H, H-4′′′), 3.88-3.89 (m, 2H, H-4′′), 4.0
(brs, 2H, H-1′′), 6.34-6.35 (m, 2H, H-3' and H-3), 6.42 (d, 1H, J = 8.0 Hz, H-5′), 7.36-
7.39 (m, 1H, H-4), 7.56 (t, 1H, J = 8.0 Hz, H-7′′′), 7.68 (d, 1H, J = 8.0 Hz, H-6′), 7.72
(brs, 1H, NH), 7.77-7.80 (m, 1H, H-5′′′), 7.86 (t, 1H, J = 8.0, H-6′′′) 8.19 (d, 1H, J = 4.0
Hz, H-6), 8.36 (d, 1H, J = 8.0 Hz, H-8′′′), 10.52 (brs, 1H, OH), 11.41 (brs, 1H, OH);
13C NMR (100 MHz, DMSO-d6): δ 20.72, 21.82, 24.30, 26.14, 27.06, 28.42, 47.15,
49.18, 103.23, 108.37, 111.88, 114.08, 115.95, 117.29, 118.96, 119.56, 125.59, 133.23,
134.20, 138.25, 139.64, 144.70, 151.02, 156.20, 161.76, 162.52, 164.17, 192.76. ESI-
MS (m/z): Calculated for C29H28N3O4 483.22, found [M+H]+ 484.46. Analysis calc. for
10

ACCEPTED MANUSCRIPT
(C28H27N3O4. 0.017 H2O): C 72.03, H 6.04, N 8.69 %; found: C 72.33, H 6.11, N
8.71%.
2.4 Inhibition studies on acetylcholinesterase
The enzymatic activity was measured using an adaptation of the method previously
described [19]. The assay solution contained 374 L of (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (Hepes) buffer (50 mM and pH 8.0), 476 L of 5,5'-
dithiobis-(2-nitrobenzoic acid) (DTNB, 3 mM), a variable volume (10-50 L) of the
stock solution of each compound in methanol (1 mg/mL), 25 L of AChE (type VI-S,
from electric eel) stock solution and the necessary amount of methanol to attain 0.925
mL of sample mixture in a 1 mL cuvette. The samples were left to incubate for 15 min
and then 75 L of acetylthiocholine iodide (AChI) solution (16 mM) was added. The
reaction was monitored for 5 min at 405 nm. Assays were run with a blank containing
all the components except AChE, which was replaced by Hepes buffer. The velocities of
the reaction were calculated as well as the enzyme activity. A control reaction was
carried out using the sample solvent (methanol) in the absence of any tested compound
and it was considered as 100% activity. The percentage inhibition of the enzyme activity
due to the presence of increasing test compound concentration was calculated by the
following Eq. (1),
(1)
in which vi is the initial reaction rate in the presence of inhibitor and vo is the initial rate
of the control reaction. The inhibition curves were obtained by plotting the percentage
of enzymatic inhibition versus inhibitor concentration and a calibration curve was
obtained from which the linear regression parameters were obtained.
2.5 DPPH radical scavenging assays
The antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging activity assays previously described [24]. To a 2.5 mL solution of
DPPH (0.002%) in methanol, four samples of each compound solution were added with
different volumes to obtain different concentrations in a 3.5 mL final volume. The
samples were incubated for 30 min at room temperature. The absorbance was measured
11

ACCEPTED MANUSCRIPT
at 517 nm against the corresponding blank (methanol). The antioxidant activity was
calculated by equation (2).
The tests were carried out in triplicate. The compound concentrations providing 50% of
antioxidant activity (EC50) were obtained by plotting the antioxidant activity against the
compound concentration.
2.6 Solution equilibrium studies
2.6.1 Potentiometric and spectrophotometric studies
pH-potentiometric and UV-vis spectrophotometric titrations of compound 6 were
°
accomplished in a 30% w/w DMSO/H2O medium, at T = 25.0  0.1 C and ionic
strength (I) 0.1 M KCl, by using 0.1 M KOH as titrant. Both glass and Ag/AgCl
reference electrodes were previously conditioned in different DMSO/H2O mixtures of
increasing DMSO % composition and the response of the glass electrode was evaluated
by strong acid – strong base (HCl/KOH) calibrations with the determination of the
Nernst parameters by Gran’s method [18]. The measurements were performed in a final
volume of 20.00 mL, the ligand concentrations (CL) were 1  10-3 M (potentiometry)
and 2–3.75  10-5 M (spectrophotometry), under different CM/CL ratios: 0:1 (L), 1:1
(M/L, M = Fe, Cu, Zn), 1:2 (M/L, M = Cu, Zn) and 1:3 (Fe/L). The spectrophotometric
measurements were carried out in a 250–500 nm wavelength range at pH ca 3–11. All
titrations were performed in triplicate and under the stated experimental conditions the
pKw value (14.4) was determined and subsequently used in the computations. The
stepwise protonation constant of the ligand, K = HL/LH, and the overall metal–

complex stability constants,
= MmHhLl/MmHhLl, were calculated by fitting
MmHhLl
the pH-potentiometric and spectrophotometric data with, respectively, Hyperquad 2008
[25] and PSEQUAD programs [26]. The Fe(III) hydrolysis model was determined under
the defined experimental conditions (I = 0.1 M KCl, 30% w/w DMSO/H2O, T = 25.0 
o


0.1 C) and the values of stability constants were log
= -6,78 and log
= -
FeH3
FeH2
10,78; the Cu(II) and Zn(II) hydrolysis constants under the same experimental



= -14.7, log
conditions (log
= -9.94; log
= -22.08 [14]) were also
Cu2H2
ZnH2
ZnH3
12

ACCEPTED MANUSCRIPT
included in the fitting of experimental data towards the equilibrium models related to
the Cu(II)/L and Zn(II)/L systems.
The species distribution curves were obtained with the Hyss program [25].
2.6.2 ESI-MS Spectra
The pH of the Fe(III)/6 (CL = 1.2  10-3 M), Cu(II)/6 (CL = 6  10-4 M) and Cu/16 (CL =
6  10-4 M) systems in aqueous solutions was previously ascertained to 4.04 and 6.01
(CL/CFe = 3) as well as to 6.81 (CL/CCu = 1 for 6), 6.30 (CL/CCu = 2 for 6), 6.00 (CL/CCu
= 1 for 16) and 6.60 (CL/CCu = 2 for 16) by using KOH solution, and the respective
mass spectra were obtained from a LCQ Fleet mass spectrometer operated in the ESI
positive and negative ion mode (Thermo Scientific). The optimized parameters were as
following: ion spray voltage, +4.5 kV; capillary voltage, 16 V; tube lens offset, -63 V;
sheath gas (N2), 80 arbitrary units; auxiliary gas, 5 arbitrary units; capillary temperature,
250 ᵒC. The spectra were recorded in the range 100 – 1000 Da. Spectra typically
correspond to the average of 20–35 scans.
3. Results and Discussion
3.1 Molecular modeling studies of TAC-HBP hybrids
Docking studies of TAC-HBP hybrids into AChE, an enzyme involved in cholinergic
loss, were performed in order to gain an insight into the type of interactions eventually
established between this enzyme and the proposed inhibitors, therefore predicting the
binding modes.
The active site of AChE contains a deep pocket where acetylcholine (substrate) can slip
inside and be hydrolyzed into acetic acid and choline, that is no longer a
neurotransmitter. The catalytic site of AChE is located near the bottom of that deep
gorge and it is formed by three aminoacids, Ser200, His440 and Glu327 (sequence
numbering of Torpedo Californica AChE, TcAChE), known as the ‘catalytic triad’ of
AChE [27]. There are still two main sites of binding for the AChE, one is located at the
lower part of the gorge, formed by three important amino acids, Phe330, Trp84 and
Glu199, and it is called as catalytic anionic site (CAS); the other, which is located at the
entrance of the gorge and is formed by Trp279, Asp72 and Tyr70, is called as peripheral
anionic site (PAS). Crystallographic structure has shown that the inhibitor tacrine binds
13

ACCEPTED MANUSCRIPT
to CAS through π-stacking (with residues Trp84 and Phe330) and hydrogen-bonding
interactions between its cyclic nitrogen and His440 [28]. While its aromatic
aminoquinoline moiety is largely responsible for the binding to AChE, the cyclohexyl
portion is related with the blocking of ACh approach to the active site.
The strategy followed herein for the design of new potential anti-AD drugs was the
coupling of two main moieties – tacrine (TAC) and hydroxybenzoyl-pyridone (HBP) -
through an alkyl spacer, which selection was aided by the screening of the envisioned
TAC-HBP hybrids against AChE. It is well recognized that compounds which are able
to interact with both CAS and PAS are good AChE inhibitors. Therefore, the length of
the alkyl spacer between the selected two moieties in the proposed inhibitors has been
decided by the docking study performed with program GOLD, v. 5.1 [29], in order to
assure that one moiety can interact with CAS while at the same time the other moiety is
able to interact with PAS and so attain maximum AChE inhibition.
The crystal structure of TcAChE complexed with an inhibitor was taken from RCSB
Protein Data Bank (PDB, entry 1ODC) [30]. This structure was chosen due to the
similarity found between its inhibitor and the herein designed ligands. In fact, the
original ligand (N-quinolin-4-yl-N´-(1,2,3,4-tetrahydroacridin-9-yl)octane-1,8-diamine)
contains a tacrine moiety connected through a long carbon chain to an amino-quinoline
group, which is structurally very similar to the HBP moiety. The docking calculations
were performed using the ASP scoring function, since this function has previously
proved to give the best docking predictions for AChE inhibitors [13,31]. For each
compound, one conformation was selected to give a good compromise between the best
consensus score and that with the closest alignment to the original ligand.
The molecular modelling studies revealed favorable interactions for three proposed
compounds (15-17, see Fig. 2), which present many similarities in their binding
conformations. Actually, Fig. 2 shows that the three designed ligands are well inserted
into the cavity of the active site, blocking the entrance to the substrate (choline) and
water molecules. The tacrine moiety is always found completely implanted close to the
bottom of the gorge of the enzyme, binding to the CAS by π–π stacking with the
aromatic ring of Trp84 and Phe330 (as in tacrine crystallographic structure), and
overlapping almost perfectly with the tacrine moiety of the original ligand. On the other
way, both the alkyl spacer and the pyridone ring seem to be well accommodated along
the hydrophobic cavity, while the benzoyl ring of the designed inhibitors is always
placed at the entrance of the gorge, being able to bind with PAS through aromatic
14

ACCEPTED MANUSCRIPT
stacking with Tyr70 and Trp279. Interestingly, compound 17, which has an alkyl spacer
of four methylene carbon atoms, establishes a further favorable H-bond between the OH
group of the benzoyl ring attached to the HBP moiety and ASP275 residue. In the case
of compounds 15 and 16, which have an alkyl spacer of two methylene units, there is no
hydrogen bond formation with ASP275, probably due to the smaller chain size but also
to the fact that these compounds contain one methoxy and one hydroxy groups instead
of two hydroxy groups as in 17. Overall, the docking into AChE of the designed
compounds (15-17) suggests their ability to interact with both the CAS and PAS,
therefore being able to act as dual-binding site AChE inhibitors, which is undoubtedly
encouraging to pursuit their synthesis and evaluation as potential multifunctional anti-
AD drugs.
Fig. 2 - Docking of TAC-HBP hybrids into AChE: superimposition of 15 (violet), 16
(light green) and 17 (pink) with original ligand N-quinolin-4-yl-N'-(1,2,3,4-
tetrahydroacridin-9-yl)octane-1,8-diamine (yellow). H-bond is represented as a solid
sky-blue line.
3.2 Synthesis of the compounds
All the designed HBP derivatives (6-8) and TAC-HBP hybrids (15-17) have been
synthesized from the (E)-ethyl-3-(4-oxo-4H-chromen-3-yl) acrylates (3-5) and coupling
of free amino alkyl substituted HBP derivatives (12-14) with 9-chloro-1,2,3,4-
tetrahydroacridine (2), respectively (Scheme). The key intermediates 3-5 were first
synthesized by following the literature procedure [16], which, from reaction with
various alkylamines and t-butyl(2-aminoethyl)carbamate in ethanol under catalytic
amount of triethylamine, gives the desired compounds (6-8) and intermediates 9-11
under a three-step addition-elimination and rearrangement sequence in one pot. The free
amino alkyl substituted HBP derivatives (12-14) were then obtained by the acid
hydrolysis of the carbamate group from 9-11. Finally, the coupling of 12-14 with 2
(synthesized from literature procedure [19]) in the presence of phenol and a catalytic
amount of potassium iodide under reflux gave the desired TAC-HBP conjugates 15-17
(Scheme).
15

ACCEPTED MANUSCRIPT
Scheme - Synthesis of HBP derivatives. Reagents and conditions: a) cyclohexanone,
o
POCl3, 180 C, 4 hr; b) R1NH2 (1.05 eq), NEt3 (1-2 drops), C2H5OH, reflux, 8-10 h; c)
DCM, TFA, 7-8 hr; d) Phenol, KI, 165-170 oC 15 min.
3.3 Acetylcholinesterase inhibitory activity
The AChE inhibitory activities of the newly synthesized TAC-HBP conjugates (15-17),
as well as of the model compound 6, were evaluated by adaptation of the spectroscopic
method described by Ellman [13]. The IC50 values for AChE inhibition are summarized
in Table 1. As expected from molecular docking, all of the three designed conjugates
display high inhibitory activities against this enzyme, with IC50 values lying in the sub-
micromolar range. Out of the three evaluated hybrids, conjugate 17 appears to be the
strongest inhibitor, with IC50 value of 0.57 µM, quite close to the value obtained for
tacrine (IC50 = 0.31 µM), followed by 16 and 15 with IC50 values of 0.71 and 0.78 µM
respectively. The HBP derivative 6 exhibited AChE inhibition at significantly higher
concentration (IC50 = 840 µM), thus demonstrating the protagonist role of the tacrine
moiety in the enzyme inhibition of the developed tacrine hybrids. Moreover, the
structure–activity analysis of these compounds allows concluding that the inhibitory
potency against AChE is closely related with the length of the linker between the two
coupled moieties. In fact, analysis of the results suggests that compound 17, containing
a four methylene unit’s linker between tacrine and HBP, seems to provide a suitable
length to fit comfortably between CAS and PAS site of enzyme, allowing the
establishment of a H-bond between an OH group of the benzoyl ring in the HBP moiety
and ASP275 residue in the PAS as already seen from the docking studies, which
explains its better AChE inhibitory activity than 15 and 16 with shorter chain lengths
and unable to form H-bonding. Although GOLD program used in the present work is
not able to determine the ligand – protein binding energy and only three experimental
points were obtained herein, it was tentatively established a correlation between the
fitness score values of GOLD, resulting from the docking of each compound (15-17),
and the experimentally obtained IC50 values. A good linear correlation (R2 = 0.9947, see
Fig. S13 in Supplementary Information) was observed, evidencing some potential
correspondence between the fitness of the compounds to the enzyme and their
respective inhibitory activity.
16

ACCEPTED MANUSCRIPT
Table 1 – Inhibitory activity (IC50, M) of AChE and DPPH radical scavenging capacity
(EC50, M) for the studied compounds, tacrine and huperzine A.
The new developed tacrine hybrids appear to have a slightly lower capacity to inhibit
AChE then the reference drugs, tacrine and the racemic mixture ()-HupA [33], but they
are expected to exhibit additional properties, such as antioxidant activity and metal
chelating capacity provided by the HBP moiety, which will make them promising
candidates for the multi-target combat of Alzheimer´s disease.
3.4 In vitro free radical scavenging activity
Antioxidants are considered valuable therapeutic tools in the prevention or therapy of
AD since there is evidence that AD brains exhibit oxidative injury in several classes of
cellular macromolecules [34].
Therefore, a series of six selected compounds, namely three HBP derivatives (6-8) and
three TAC-HBP conjugates (15-17), was herein screened for their DPPH radical
scavenging capacity (EC50) [24] and the corresponding results are summarized in Table
1. Analysis of the experimental data obtained shows that, among the HBP derivatives
(6-8), the 2′,5′-dihydroxybenzoyl substituted compound 8 was found to exhibit an
exceptional good antioxidant activity (EC50 = 4.28 µM), even better than the value
found by the same method for vitamin C (EC50 = 15 µM [35]), followed by the 2′,4′-
dihydroxybenzoyl substituted derivative 7 with EC50 value of 95 µM. Interestingly,
although both compounds 7 and 8 carry the same number of hydroxyl groups (two) on
the benzoyl ring, they show a huge difference in antioxidant activity. The much higher
activity of compound 8 in comparison with 7 can be explained by the fact that the
former is able to release hydrogen for DPPH quenching more easily than the later one
due to the possible formation of a stable 1,4-quinone. Furthermore, as already expected,
the substitution of one hydroxyl group of 7 by a methoxy unity (compound 6) is
responsible for an accentuated decrease (ca 2-fold) in radical scavenging capacity.
The analysis of the DPPH radical scavenging activity data for the TAC-HBP conjugates
(15-17) shows that even though their activity is lower than that of the HBP compounds,
the found DPPH radical scavenging capacity is much improved when compared with
that of the parent drug tacrine (EC50  1000 M). Moreover, the radical scavenging
17

ACCEPTED MANUSCRIPT
activity screening of these TAC-HBP conjugates suggests that the 2′,4′-dihydroxy
substituted conjugate 17 exhibits a higher potential (EC50 = 204 µM) than the 2′-
hydroxy -4′/5′-methoxy substituted conjugates 15-16 (EC50 213-249 µM), according
with the results obtained for the HBP compounds. So, as a conclusion, it can be stated
that HBP moieties are successful in bringing antioxidant activity to hybrids 15-17 which
have been synthesized from a non-active tacrine.
3.5 Metal chelating capacity
Research on neurodegenerative diseases has also been dealing with metal chelation, due
to the general recognition that chelators interfere not only in metal-induced Aβ
aggregation and neurotoxicity but also on metal homeostasis of AD brains. Since the
herein studied tacrine derivatives 15-17 have a common 2-hydroxybenzoyl chelating
core, compound 6, a 2′-hydroxy-4′-methoxybenzoyl-2-pyridinone derivative, was
chosen as a model to analyze their chelating capacity towards redox-active (Fe(III),
Cu(II)) and Aβ-binding (Cu(II), Zn(II)) metal ions. Due to solubility reasons, these
equilibrium solution studies were accomplished in a mixed 30% (w/w) DMSO/water
medium, In fact, DMSO was chosen because it is a quite used and well tolerated solvent
in biological and cellular studies, the amounts of ligand ( 7 M) and DMSO ( 1%)
employed in culture media being quite low with concomitant no alterations observed in
cells [13,36].
Firstly, the protonation constant (log K) of 6, corresponding to the phenolic hydroxyl
group, was determined by pH-potentiometric as well as ultraviolet-visible (UV-vis)
spectrophotometric titrations (see Fig. 3) reaching identical values (see Table 2).
Nevertheless, in the following calculations for the metal/6 systems, the log K value
obtained by each experimental method will be used in the complexation studies
performed by the same methodology.
Fig. 3 - a) pH-potentiometric titration curves of 6 (CL = 1  10-3 M); b)
Spectrophotometric absorption spectra of 6 recorded for 3.68 < pH < 11.05 (CL = 2 
10-5 M) and inset containing the individual calculated spectra by PSEQUAD.
18

ACCEPTED MANUSCRIPT
Table 2 - Protonation constant of model compound 6, global formation constantsa of its
º
metal (Fe(III), Cu(II), Zn(II)) complexes (T = 25.0  0.1 C, I = 0.1 M KCl, 30% w/w
DMSO/water) and pMb values as well as for other selected bidentate chelators.
Compound 6 was obtained in the neutral monoprotonated form (HL). The inset of Fig. 3
b), containing the calculated spectra (PSEQUAD) of the mono- and deprotonated forms
of this compound, as well as Fig. 4, prove that the maximum at 297 nm corresponds to
both species (HL and L) while that at 383 nm belongs only to the deprotonated L form.
Fig. 4 - Species distribution curves for 6 with molar extinction coefficients at the
maximum absorption wavelengths (CL = 2  10-5 M).
Although compound 6 contains a hydrophilic OH group, at the physiological pH (7.4)
and for CL = 10-5 M it is mainly in the neutral HL form (97.5%), thus reflecting a low
hydrophilicity and the need to use a 30% DMSO/water medium in solution studies. The
high value of the protonation constant of 6 (ca 9) reflects the stabilization of the
protonated HL species resulting from the establishment of an intramolecular H-bond
between the phenolic hydroxide (H-bond donor) and the O-ketone (H-bond acceptor).
Besides stabilizing the enol form of the phenol group, there is also a conjugation effect
with the double bond of the ketone group. In fact, this has also been observed (in
aqueous medium) for 2-acetylphenol (log K = 9.87) when compared to its analogue 4-
acetylphenol (log K = 8.05) [37].
The chelating ability of compound 6 towards the three metal ions under study – Fe(III),
Cu(II) and Zn(II) - was evaluated through the determination of the global formation
constants of the complexes by pH-potentiometric (Hyperquad 2008 program [24])
titrations and also for the Fe(III) complexes by UV-vis spectrophotometry (PSEQUAD
program [25]). From Fig. 2 a) it is possible to observe that for all the metal ions under
study there is a large change in the deprotonation profile of the ligand titration curve
due to the presence of the metal ion. Actually, all the curves of the metal/6 systems lye
below that of the ligand for 0  a  1, therefore evidencing the formation of metal
complexes with the deprotonated form of the ligand (L) with relative stability order Fe 
Cu  Zn. These results point towards a β-phenol-keto coordination mode, with
19

ACCEPTED MANUSCRIPT
formation of 6-membered rings, such as already found for the Co(II), Ni(II) and Cu(II)
complexes of salicylaldehyde [43].
As stated above for the Fe(III)/6 system, both pH-potentiometric and
spectrophotometric titrations were performed for the 1:1 and 1:3 Fe(III)/ligand molar
ratios, although the complexation model presented in Table 2 and used in Fig. 5 a) for
the 1:3 system corresponds to the one obtained by spectrophotometry since at the
beginning of potentiometric titration (CL = 1  10-3 M) both FeL and FeL2 coexisted
with ca 80% FeL.
Fig. 5 – a) Species distribution curves for the 1:3 Fe(III)/6 system with molar extinction
coefficients at the maximum absorption wavelengths (CL = 3.75  10-5 M); b)
Spectrophotometric absorption spectra of 6 (pH = 7.80, CL = 2  10-5 M) and Fe(III)/6
1:3 (pH = 6.44, CL = 3.75  10-5 M).
From the spectra obtained for the 1:1 Fe(III)/6 system, βFeL was calculated and the
stability constants of the remaining complex species were determined from the titration
performed under 1:3 molar stoichiometry of metal ion to ligand while holding the value
of βFeL constant. Fig. 5 a), containing the species distribution curves for the 1:3 Fe(III)/6
system, shows that at the used experimental conditions, the iron complexation begins
below pH 3, with the formation of both FeL and FeL2 complexes, while FeL3 is
predominant above pH ca 4.5 (78% of FeL3 at pH 7.4 and micromolar metal
concentration - CL = 10 × CM), therefore confirming the expected good chelating power
of compound 6 toward Fe(III).
All the iron complex species formed have identical absorption maximum at ca 296 nm
up to pH 8, but above that value another band appears at 386 nm corresponding to the
deprotonated ligand (Fig. 5 a)). The superposition of UV-vis spectra of Fe(III)/6 (pH =
6.44) and 6 (see Fig. 5 b)) reveals that the coordination of the Fe(III) ion is responsible
for an high increase in the intensity of the band at 296 nm for the complex although no
shift of this band is observed.
Both studies of Cu(II) and Zn(II) complexation with compound 6 were performed by
potentiometric titration and the respective species distribution curves for the 1:2
Cu(II)/6 and 1:1 Zn(II)/6 systems at the used experimental conditions are shown in
Fig.5. For the 1:1 and 1:2 Cu(II)/6 systems precipitation occurred above ca pH 6,
20

ACCEPTED MANUSCRIPT
probably due to the formation of insoluble hydroxocomplexes, while for the Zn(II)/6
systems no precipitation was observed. It is also possible to see that, at the
concentrations used in the experimental conditions, complexation of Cu(II) and Zn(II)
begins above pH 4 and 5.5, respectively (see Fig. 6), and that a strong competition
exists with metal hydroxide species.
Fig. 6 - a) Species distribution curves for the 1:2 Cu(II)/6 and b) 1:1 Zn(II)/6 systems
(CL = 1  10-3 M).
ESI-MS data was also obtained for solutions of Fe(III)/6 and Cu(II)/6 in 1:3 (Fe/L), 1:1
(Cu/L) and 1:2 (Cu/L) stoichiometric conditions and at convenient pH values (see
Experimental part). These spectra confirmed the existence of 1:1 and 1:3 iron
complexes, respectively, as [Fe(HL)Cl2]+, [FeL(HL)2Cl]+ and [FeL3Cl]-; regarding the
copper complexes, 1:1 species, such as [Cu(HL)Cl]+, as well as polymeric species
[Cu2L3]+ were found, the last one in a higher quantity for the 1:2 metal/ligand molar
ratio stoichiometric conditions (see Table 3). Under the experimental conditions of
ESI/MS it was not possible to observe the formation of the CuL2 complex, possibly
owing to its low percent formation and also to some precipitation of hydroxo species.
Table 3 - ESI-MS ions for the Fe(III) and Cu(II) complexes of 6 in aqueous solution
(1:3 Fe/L, CL = 1.2  10-3 M; 1:1 and 1:2 Cu/L, CL = 6  10-4 M).
Comparison of the metal chelating capacity of compound 6 with that of some selected
bidentate analogous chelators (see Table 2), namely salicylaldehyde and
2-
acetylphenol, allows to conclude that the values of the corresponding global stability
constants tend to be analogous, even though different solution media and ionic strengths
were used. In fact, it is not easy to establish an accurate comparison since, in some
cases, pM values in Table 2 were determined by using different metal hydrolysis models
(2-acetylphenol) or were not determined (salicylaldehyde with I = 0.5 M), while in other
cases (salicylaldehyde, 2-acetylphenol) the model for the Fe(III)/L systems was
incomplete for the medium solution conditions chosen with corresponding decrease of
pFe values. Since precipitation occurred above pH 6 in the case of Cu(II)/6 systems,
that pH value was chosen to determine all the pM values corresponding to compound 6
(see Table 2). Nevertheless, when comparing the calculated pFe and pZn values for 6 at
21

ACCEPTED MANUSCRIPT
pH 7.4 (pFe = 18.1, pZn = 6.5), to simulate physiological conditions, with those of a
well known strong iron chelator, deferiprone (DFP), at the same pH value (pFe = 19.4 -
20.7, pZn = 6.2) it is possible to infer that compound 6 is a good iron chelator, although
weaker than DFP. These values reflect two features working in opposite direction: the
fact that 6-membered chelate rings are involved in the complexes of 6, against more
favorable 5-membered ones in those of DFP; also the fact that a 30% w/w DMSO/H2O
medium can increase the values of stability constants relative to an aqueous medium,
due to the relative balance established between electrostatic and non electrostatic forces
for media with different dielectric constants [44].
Besides evaluating the chelating capacity of the HBP moiety with model compound 6,
the chelating ability of the TAC-HBP hybrids was also confirmed by UV-vis
spectrophotometry and ESI-MS spectrometry with compound 16. Actually, Fig 7 shows
an increase in the intensity of the UV bands when copper is coordinated to compound
16, similarly to previously observed for 6 and Fe(III) (see Fig. 5b)); also, the presence
of 1:2 (Cu/L) complexes, as [CuL2H]+ and [Cu(HL)2Cl]+ (see Table 3), was confirmed
by ESI-MS. Unfortunately, for the system Cu(II)/16, under the experimental conditions
of ESI/MS, it was not possible to observe the formation of the CuL complex.
Fig. 7 – a) Spectrophotometric absorption spectra of 16 and Cu(II)/16 1:2 (pH = 7.40,
CL = 2  10-5 M); b) Expanded ESI(+) mass spectrum of Cu(II)/16 1:1 (pH=6.00, C =
L
6  10-4 M) showing a group of peaks at m/z 999.8 /1001.9 attributed to [CuL2H]+
species. The inset presents the calculated isotopic pattern for the cation
[C56H53CuN6O8]+.
In this section it has been proved that 5-(2-hydroxybenzoyl)-pyridin-2(1H)one moieties
are moderate/good chelators of Fe(III), Cu(II) and Zn(II) and that by including them in
the design of potential anti-AD drugs their chelating capacity towards that well-known
triad of transition metal ions can be assured.
3.6. Molecular modeling of the 1:3 Fe(III)/6 complex
In the absence of X-ray structures for the metal complexes of model compound 6, a
molecular modelling study was accomplished to show the 1:3 Fe(III)/6 iron complex
22

ACCEPTED MANUSCRIPT
structure. This study was carried out with full geometry optimization of the ligand and
respective Fe(III) complex by quantum mechanical calculations based on density
functional theory (DFT) methods [45] included in the Gaussian 03 program software
[46] with the B3PW91 functional [47]. Geometry optimizations were obtained by using
3-21G basis set [48] for all elements, first, and afterwards re-optimized using 6-31G**
[49] for the main group elements and the Stuttgart–Dresden pseudopotential (SDD) and
associated basis set for Fe [50]. No symmetry constrains were enforced during geometry
optimizations.
Fig. 8 – DFT-minimized structure of the FeL3 complex of compound 6. Coloring of
atoms: Fe turquoise, N blue, O red, C grey and H white.
The energy-minimized structure obtained for complex FeL3 of compound 6 confirms a
β-phenol-keto metal coordination core for each ligand, with a slightly distorted
octahedral geometry entirely composed by hard oxygen donor atoms (see Fig. 8). In
fact, coordination bond distances are somehow higher for Fe-Oketo (1.925-1.946 Å)
than for Fe-Ophenol (1.871-1.896 Å) and also bond angles involving the hard iron metal
ion (O-Fe-O) tend to be slightly higher within the hexadentate chelating ring of each
0
0
ligand (89.0-92.2 C) than between different ligands (89.4-90.2 C). These parameters
reveal that the keto oxygen atoms are less strained than the phenolic ones therefore
compensating the relatively more rigid phenolic donor atoms and allowing a good
accommodation of the metal ion in the coordination shell.
1. Conclusions
Tacrine-(hydroxybenzoyl-pyridone) (TAC-HBP) hybrids were designed by conjugating
an active AChE inhibitor (tacrine) with a 2-hydroxybenzoyl-2-pyridone (HBP) moiety
through an alkyl chain with adequate length capable to assure dual-binding mode of
enzyme inhibition. After preliminary docking studies, three TAC-HBP conjugates (15-
17) were selected to be synthesized, biologically evaluated for AChE inhibition and also
assayed for radical scavenging activity (DPPH method). Screening results showed that
all the compounds display inhibitory activities close to tacrine, with IC50 values lying in
the sub-micromolar range (0.57-0.78 M), and exhibit good antioxidant activity (EC50 =
204-249 M) conferred by the HBP moiety. The higher anti-AChE activity of
23

ACCEPTED MANUSCRIPT
compound 17 (IC50 = 0.57 M) confirms the possible better fitting of this compound
between CAS and PAS as well as its capacity for H-bonding as previewed by molecular
modelling studies. Finally, the chelating capacity of the developed tacrine hybrids was
evaluated by studying the complexation ability of a 2′-hydroxy-4′-methoxybenzoyl-2-
pyridinone derivative (compound 6), as a model compound, towards three redox-active
and/or Aβ-binding metal ions (Fe(III), Cu(II)), Zn(II)) by using potentiometric, UV-vis
spectrophotometric and ESI-MS techniques. It was proved that the HBP moiety acts as a
moderate/good chelator of these metal ions (pFe = 13.9, pCu = 6.0 and pZn = 6.0 at pH
6.0, CL/CM = 10, CM = 10-6 M), being able to form complexes of variable maximum
metal/ligand stoichiometry: 1:3 for Fe(III), 1:2 for Cu(II) and 1:1 for Zn(II). Molecular
modelling of the 1:3 iron complex of model compound 6 showed an octahedral
coordination around the iron(III) metal ion, involving a β-phenol-keto coordination
mode. UV and ESI-MS spectra for the system Cu(II)/16 proved that the hybrids are also
able to chelate metal ions. In the search for multifunctional anti-Alzheimer’s disease
drugs, the performed work evidenced the importance of conjugating these two main
moieties. In fact, the resulting tacrine conjugates are dual-binding site AChE inhibitors,
with anti-AChE activity comparable to the parent compound tacrine, but further
showing antioxidant capacity and biometal chelation ability conferred by the HBT
moiety.
24

ACCEPTED MANUSCRIPT
Abbreviations
Aβ
amyloid-β
ACh
acetylcholine
AChE
AChI
AD
acetylcholinesterase
acetylthiocholine iodide
Alzheimer´s disease
BuChE
CAS
butyrylcholinesterase
catalytic anionic site
ChEIs
COSY
DCM
DFP
cholinesterase inhibitors
correlation spectroscopy
dichloromethyl
deferiprone
DFT
density functional theory
deoxyribonucleic acid
DNA
DPPH
DTNB
ESI-MS
HBP
Hepes
HSQC
HupA
MTDLs
PAS
2,2-diphenyl-1-picrylhydrazyl
5,5'-dithiobis-(2-nitrobenzoic acid)
electrospray ionization-mass spectrometry
hydroxybenzoyl-pyridone
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
heteronuclear single quantum coherence spectroscopy
Huperzine A
multi-target-directed ligands
peripheral anionic site
ROS
RT
reactive oxygen species
room temperature
SDD
TAC
Stuttgart–Dresden pseudopotential
tacrine
TFA
trifluoracetic acid
TLC
UV-vis
thin layer chromatography
ultraviolet-visible spectrophotometry
25

ACCEPTED MANUSCRIPT
Acknowledgments
The authors thank the Portuguese NMR (IST-UL Center) and Mass Spectrometry
Networks (Node IST-CTN) for providing access to their facilities, as well as to Dr.
Maria da Conceição Oliveira for helping in the interpretation of ESI-MS data. Financial
support is acknowledged to the Portuguese Foundation of Science and Technology
(FCT) for Project UID/QUO/ 00100/2013 and fellowship to K.C. H.M.A. is thankful to
Erasmus Mundus ACTION2 Program (WELCOME Scholarship WELC1104289).
26

ACCEPTED MANUSCRIPT
References
[1] R. Jakob-Roene, H. Jacobsen, Angew. Chem. Int. Ed. 48 (2009) 3030-3059.
[2] E. Scarpini, P. Scheltens, H. Feldman, Lancet Neurol. 2 (2003) 539-547.
[3] L.M. Sayre, G. Perry, M.A. Smith, Chem. Res. Toxicol., 21 (2008) 172–188.
[4] C.G. Ballard, Eur. Neurol. 47 (2002) 64-70.
[5] E. Giacobini, Neurochem. Int. 32 (1998) 413-419.
[6] R. León, A.G. Garcia, J. Marco-Contelles, Med. Res. Rev., 33 (2013) 139-189.
[7] A. Nunomura, G. Perry, G. Aliev, K. Hirai, A. Takeda, E.K. Balraj, P. K. Jones, H.
Ghanbari, T. Wataya, S. Shimohama, S. Chiba, C.S. Atwood, R.B. Petersen, M.A.
Smith, J. Neuropathol. Exp. Neurol. 60 (2001) 759-767.
[8] X.D. Huang, R.D. Moir, R.E. Tanzi, A.I. Bush, J.T. Rogers, Ann. N. Y. Acad. Sci.
1012 (2004) 153-163.
[9] A.P. Kozikowski, G. Campiani, L.-Q. Sun, S. Wang, A. Saxena, B.P. Doctor, J. Am.
Chem. Soc. 118 (1996) 11357-11362.
[10] X. Sui, C. Gao, Int. J. Mol. Med. 33 (2014) 227-233
[11] M.E. Wall, Med. Res. Rev. 18 (1998) 299-314.
[12] M. Torres, S. Gil, M. Parra, Curr. Org. Chem. 9 (2005) 17-54757-1779.
[13] A. Nunes, S.M. Marques, C. Quintanova, D.F. Silva, S.M. Cardoso, S. Chaves,
M.A. Santos, Dalton Trans 42 (2013) 6058-6073.
[14] C. Quintanova, R.S. Keri, S. Chaves, M.A. Santos, J. Inorg. Biochem. 151 (2015)
58-66.
[15] R.S. Keri, C. Quintanova, S. Chaves, D.F. Silva, S.M. Cardoso, M.A. Santos,
Chem. Biol. Drug Des. 87 (2016) 101-111.
[16] C. Karam, S. Prasad, K. Rakesh, R.K. Tiwari, A.N. Shirazi, S. Kumar, K. Parang,
S.K. Sharma, Bioorg. Chem. 53 (2014) 75-82.
[17] W.L.F. Armarego, D.D. Perring (Eds) Purification of laboratory chemicals,
Butterworth–Heinemann Press, 4th ed, Oxford, 1999.
[18] F.J.C. Rossotti, H. Rossotti, J. Chem. Ed. 42 (1965) 375-378.
[19] R.S. Keri, C. Quintanova, S.M. Marques, S.M. Cardoso, M.A. Santos, Bioorg.
Med. Chem. 21 (2013) 4559-4569.
[20] Maestro, version 9.3. Schrödinger Inc.: Portland, OR, 2012.
[21] T. Hassinen, M. Peräkylä, J. Comput. Chem. 22 (2001) 1229-1242.
27

ACCEPTED MANUSCRIPT
[22] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol. 267 (1997)
727-748.
[23] K. Chand, A.K. Sharma, S.K. Sharma, Magn. Reson. Chem. 54 (2016) 91-102.
[24] B. Tepe, D. Daferera, A. Sokmen, M. Sokmen, M. Polissiou, Food Chem. 90
(2005) 333-340.
[25] P. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 1739-1753.
[26] L. Zékány, I. Nagypál, G. Peintler, PSEQUAD version 5.01, 2001.
[27] H. Dvir, I. Silman, M. Harel, T.L. Rosenberry, J.L. Sussman, Chem-Biol. Interact.
187 (2010) 10–22.
[28] M. Harel, I. Schalk, L. Ehret-Sabatier, F. Bouet, M. Goeldner, C. Hirth, P.H.
Axelsen, I. Silman, J.L. Sussman, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 9031-
9035.
[29] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol., 267, (1997)
727-748.
[30] Protein Data Base (PDB). http://www.pdb.org/pdb/home/home.do
[31] J. Sebestik, S.M. Marques, P.L. Fale, S. Santos, D.M. Arduino, S.M. Cardoso, C.R.
Oliveira, M.L. Serralheiro, M.A. Santos, J. Enzyme Inhib. Med. Chem. 26 (2011) 485-
497.
[32] X. Chao, X. He, Y. Yang, X. Zhou, M. Jin, S. Liu, Z. Cheng, P. Liu, Y. Wang, J. Yu,
Y. Tan, Y. Huang, J. Qin, S. Rapposelli, R. Pi, Bioorg. Med. Chem. Lett. 22 (2012)
6498–6502.
[33] X.C. Tang, G.H. Kindel, A.P. Kozikowski, I. Hanin, J. Ethnopharmacol. 44 (1994)
147–155.
[34] D. Pratico, Trends Pharmacol. Sci. 29 (2008) 609-615.
[35] Y. Zhao, J. Dou, T. Wu, H.A. Aisa, Molecules,18 (2013) 951-962.
[36] S.M. Marques, C.C. Abate, S. Chaves, F. Marques, I. Santos, E. Nuti, A. Rossello,
M.A. Santos, J. Inorg. Biochem. 127 (2013) 188–202.
[37] A.E. Martell, R.M. Smith, Critical Stability Constants, Vol. 3, Plenum Press, New
York and London, 1977.
[38] R.M. Smith, A.E. Martell, Critical Stability Constants, Vol. 4, Plenum Press, New
York and London, 1976.
[39] Z.D. Liu, R.C. Hider, Coord. Chem. Rev. 232 (2002) 151-171.
[40] V.M. Nurchi, G. Crisponi, T. Pivetta, M. Donatoni, M. Remelli, J. Inorg. Biochem.
102 (2008) 684-692.
28

ACCEPTED MANUSCRIPT
[41] E.T. Clarke, A.E. Martell, Inorg. Chim. Acta 191 (1992) 57-63.
[42] M.A. Santos, S.M. Marques, S. Chaves, Coord. Chem. Rev. 256 (2012) 240-259.
[43] G.C. Percy, D.A. Thomton, J. Inorg. Nucl. Chem. 35 (1973) 2719-2726.
[44] S. Raju, K.B.K. Naik, B.A. Kumar, G.N. Rao, J. Ind. Chem. Soc. 89 (2012) 57-62.
[45] R.G. Parr, W. Yang, Density Functional Theory of Atoms and Molecules, Oxford
University Press, New York, 1989.
[46] Gaussian 03, Revision C.02, M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E.
Scuseria, M.A. Robb, J.R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K.N. Kudin,
J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene,
X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J Jaramillo, R.
Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G.
Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D.
Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford,
J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L
Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M.
Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A.
Pople, Gaussian, Inc., Wallingford CT, 2004.
[47] J.P. Perdew, K. Burke, Y. Wang, Phys. Rev. B 54 (1996) 16533-16539.
[48] K.D. Dobbs, W.J. Hehre, J. Comp. Chem. 8 (1987) 861-879.
[49] P.C. Hariharan, J.A. Pople, Theor. Chim. Acta 28 (1973) 213-222.
[50] T. Leininger, A. Nicklass, H. Stoll, M. Dolg, P. Schwerdtfeger, J. Chem. Phys. 105
(1996) 1052–1059.
29