Design, synthesis and evaluation of multifunctional salphen derivatives for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2014.10.004

A series of salphen derivatives (1-26) have been designed, synthesized, and evaluated as chemical reagents that target and modulate multiple facets of Alzheimer's disease. Most of the compounds exhibit a significant ability to inhibit self-induced and Cu<

Full text of DOI:10.1016/j.ejmech.2014.10.004

European Journal of Medicinal Chemistry 87 (2014) 540e551
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of multifunctional salphen
derivatives for the treatment of Alzheimer's disease
Neng Jiang, Su-Yi Li, Sai-Sai Xie, Zhong-Rui Li, Kelvin D.G. Wang, Xiao-Bing Wang*,
Ling-Yi Kong*
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
210009, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of salphen derivatives (1e26) have been designed, synthesized, and evaluated as chemical re-
Received 8 July 2014
Received in revised form
28 September 2014
Accepted 1 October 2014
Available online 2 October 2014
agents that target and modulate multiple facets of Alzheimer's disease. Most of the compounds exhibit a
significant ability to inhibit self-induced and Cu2þ-induced
b-amyloid (Ab1e42) aggregation, and to
function as potential antioxidants and biometal chelators. In particular, the antioxidant activity of
compound 2 is 2.6-fold of the trolox value by using the ABTS radical scavenging method, and it also
shows a significant ability to inhibit self-induced and Cu2þ-induced
(70.3%, 20 mM and 85.7%, 50 m
b
-amyloid (Ab1e42) aggregation
M, respectively). Moreover, it is capable of decreasing reactive oxygen
species (ROS) induced by Cu2þ-A
, shows a good neuroprotective effect in human SH-SY5Y neuroblas-
toma cells and can cross the bloodebrain barrier. In addition, compound 2 retains the activities of
antioxidant, anti A
aggregation and neuroprotection after capturing the metal ions Cu2þ, Fe3þ and Zn2þ
Keywords:
Alzheimer's disease
Antioxidant
b
b
b-Amyloid aggregation
(its metal complexes 18, 22 and 23). Taken together, these results suggest that compound 2 might be a
promising lead compound for AD treatment.
Metal chelator
Neuroprotection
The bloodebrain barrier
© 2014 Published by Elsevier Masson SAS.
1. Introduction
determine their contribution to AD neuropathogenesis [10e19].
Several findings have suggested that two avenues for metaleA
b
Alzheimer's disease (AD) is a dreadful neurological illness
characterized by a loss of brain function, affecting memory,
cognition, and behavior [1,2]. To date there is no treatment for AD
and its diagnosis with high accuracy requires a detailed post-
mortem examination of the brain [3]. The “amyloid hypothesis”
of AD states the brains of AD patients are characterized by the
deposition of amyloid plaques whose main component is the am-
species are linked to neurotoxicity: the facilitation of A aggrega-
tion and the induction of oxidative stress through ROS generation,
leading to neuronal cell death and cognitive impairment. The
b
neurotoxic effects that could be induced by metaleAb species may
be related to the disruption of membranes leading to apoptosis
and/or aggregate accumulation at and around the synapse resulting
in weakened cell signaling and neuronal death [10]. The second
avenue of neurotoxicity is associated with oxidative stress. When
yloid
cursor protein (APP) by
accumulation of A is accompanied by oxidative stress and
inflammation, which leads to neurodegeneration. Therefore, pre-
vention of A aggregation in the brain is currently being considered
as a potential therapy for AD.
shows high affinity for metal ions such as Zn2þ and Cu2þ
which are able to accelerate the formation of A aggregates and
species have been studied to
b
(Ab
) peptide generated via cleavage of the amyloid pre-
b
- and -secretases [4e9]. The progressive
g
metal ions are surrounded by A
peroxide (H2O2) and hydroxyl radicals (ꢀOH) can be generated.
Cu(I)eA oligomers have been suggested to contain a highly O2
reactive Cu(I) center, which may also assist in the formation of ROS
[20]. Overall, metal-induced A aggregation and oxidative stress
b species, ROS such as hydrogen
b
b
b
b
A
b
could be attributed to a high degree of neurotoxicity leading to
neuronal death and ultimately, cognitive impairment. Thus, the
b
neurofibrillary tangles. MetaleA
b
modulation of metaleA
of neuronal cells from oxidative damage becomes a promising way
to inhibit the A aggregation and thereby reduce the neurotoxicity
of A deposits [21].
b interaction and the successful protection
b
b
* Corresponding authors.
E-mail addresses: xbwang@cpu.edu.cn (X.-B. Wang), cpu_lykong@126.com,
lykong@cpu.edu.cn (L.-Y. Kong).
Resveratrol (trans-3,4,5-trihydroxystilbene), a phytoalexin with
a stilbene structure is present in medicinal plants, grape skin,
http://dx.doi.org/10.1016/j.ejmech.2014.10.004
0223-5234/© 2014 Published by Elsevier Masson SAS.

N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
541
peanuts, and red wine. It was shown that resveratrol could coun-
teract A toxicity by its antioxidant properties in cellular models
[22], and also could inhibit A
aggregation in vitro [23]. 18F-labeled
ML. We investigated whether salphen derivative complexes, after
b
capturing metal ion like Cu2þ, Fe3þ and Zn2þ, could retain the ac-
b
tivities of anti Ab aggregation, antioxidant, and neuroprotection,
stilbene derivatives have been developed as PET radiotracers for
imaging amyloid plagues in the brain, which implies the stilbene
and continue to play a role in Alzheimer's disease. Their corre-
sponding metal complexes have been designed to accomplish this.
In this paper, we described the design, synthesis and pharma-
cological evaluation of a series of salphen derivatives and their
metal complexes as multifunctional anti-AD agents. The pharma-
cological evaluations of these compounds include antioxidant, self-
structure has a strong affinity to A
dimethyl-N4-(pyridin-2-ylmethylene)benzene-1,4-diamine
Fig. 1), can modulate metal-induced A aggregation and ROS pro-
duction, leading to reduction of metaleA neurotoxicity in living
b
aggregates [24,25]. (E)-N1,N1-
(L1,
b
b
cells [26]. The metal chelator clioquinol (CQ) has moved into clin-
ical trials and showed improved cognition. Long-term use is,
however, limited by an adverse side effect, subacute myelo-optic
neuropathy [27]. Although these metal chelators have an adverse
side effect, some studies using these compounds show the
involvement of metal ions in AD pathogenesis [28].
induced and Cu2þ-induced
A
b
aggregation, ROS induced by
Cu2þeA
b, metal chelation, neuroprotection and the bloodebrain
barrier.
2. Result and discussion
Very recently, our group has reported the synthesis of tacri-
neecoumarin hybrids and (E)-N-benzylideneaniline derivatives as
multi-targeted agents against AD [29e31]. To develop a chemical
tool capable of both targeting and modulating the reactivity of
multiple AD pathological factors in biological systems, we designed
a novel multifunctional ligand (ML) with the potential for inter-
2.1. Chemistry
The (N1E,N2E)-N1,N2-dibenzylidenebenzene-1,2-diamine de-
rivatives 1e15 were synthesized as previously described [35]. The
commercially available 1,2-phenylenediamine derivatives were
reacted in ethanol with two equivalents of the respective salicy-
laldehyde derivatives under reflux condition (Scheme 1). Structures
of all synthesized compounds were characterized by comparison
with 1H and 13C nuclear magnetic resonance (NMR) spectroscopy
data reported in the literature. The salphen metal complexes 16e26
were synthesized by reacting the respective salicylaldehyde with
the corresponding diamine in the presence of either FeCl3$6H2O,
Cu(OAc)2$H2O or Zn(OAc)2$2H2O under reflux condition (Scheme
1).
action with A
tion and antioxidant activity (Fig. 1). For A
and metal chelation, ML was constructed by combining Resveratrol,
a known A imaging agent, with L1, a molecule previously reported
to target and regulate metaleA and CQ, a metal chelator (Fig. 1).
b
, metaleA
b, metal chelation, control of ROS genera-
b
/metaleA interactions
b
b
b
For enhanced metal binding properties, (N1E, N2E)-N1,N2-dibenzy-
lidenebenzene-1,2-diamine, along with oxygen donor, was chosen
as basic structure of bifunctional small molecules affording a tet-
radentate ligand for Cu(II) with 1:1 metal-to-ligand stoichiometry.
For antioxidant activity, substituents (i.e., phenolic groups, Fig. 1)
[32e34] known to have antioxidant capability were integrated into
The paramagnetism of the iron and copper complexes made a
characterization by NMR spectroscopy impossible. However, the
coordination of the ligands to iron(III) and copper(II) could be
Fig. 1. Combination of the main features of resveratrol, L1 and clioquinol provides molecules with multifunctionality (metal chelation, Ab interaction, and antioxidant).

542
N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
Scheme 1. Structure scheme of salphen derivatives and chemical structure of compounds investigated.
verified by some measurements (IR, Mass and Elemental analyses).
In the IR spectra of iron and copper complexes, new bands arise in
the region between 600 and 400 cmꢁ1 vibration bands of the FeeN
(v z 540 cmꢁ1), the FeeO (v ¼ 470 cmꢁ1), CueN (v z 546 cmꢁ1),
the CueO (v ¼ 427 cmꢁ1) [34]. The others were elucidated by
spectroscopic measurements (IR, Mass, 1H NMR and 13C NMR). The
IR spectra of titled compounds (1e26) showed absorption bands of
skelton vibrations for benzene rings at 1480e1590 cmꢁ1. The
characteristic strong bands appeared for C]N stretching at
1596e1637 cmꢁ1. In the 1H NMR spectra, aromatic protons
Table 1
DPPH scavenging activities and inhibitions of A
salphen derivatives.
b(1e42) self-induced aggregation of
Compounds
IC50
(
m
M)
Ab(1e42) aggregation
inhibition (%)b
DPPH scavenging activitiesa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
233 5.1
21 2.0
144 3.4
135 0.8
338 1.4
483 1.8
322 2.5
>2000
260 7.7
233 4.0
19 1.3
563 5.7
241 4.4
169 3.0
356 2.8
296 3.6
>2000
17 0.8
193 5.9
727 7.4
483 4.6
27 2.1
16 1.5
130 3.9
147 4.3
398 1.5
109.4 3.8
e
52.2 2.6
70.3 2.1
66.4 1.0
70.4 1.7
65.0 0.8
73.1 1.6
54.6 0.9
33.7 1.4
35.8 2.1
42.9 2.8
59.0 1.6
57.8 2.0
69.9 1.1
65.5 1.5
27.4 2.3
65.5 1.2
62.1 1.7
71.8 2.2
25.5 0.9
33.5 1.6
64.7 0.9
69.6 1.7
67.5 2.2
60.4 1.1
42.6 2.0
37.0 1.3
63.7 2.3
56.9 1.7
appeared as set of multiplet in the region
CH]N protons resonated as a singlet between
Moreover, in the 13C NMR spectra, the carbon resonance fre-
quencies of the CH]N was at 162.04e175.20 ppm. The aromatic
carbons appeared at 102.81e164.57 ppm. Finally, the eOCH3
groups appeared at 52.64e56.14 ppm.
d
5.22e8.54 ppm and
d
8.43 and 9.70 ppm.
d
d
d
2.2. Radical-scavenging activity
DPPH (diphenyl-1-picrylhydrazyl) radicals can be used in pre-
liminary screening of compounds capable of scavenging reactive
oxygen species, since these nitrogen radicals are much more stable
and easier to handle than oxygen free radicals [34]. For comparison
purpose, resveratrol was used as reference compound. The IC50
values of all tested compounds are summarized in Table 1. From the
table, it could be seen that compounds 2,11,18, 22 and 23 had much
better scavenging activities than resveratrol, while compounds 3, 4,
10, 13, 14, 19, 24 and 25 had similar radical scavenging activities
compared to resveratrol. These results indicated that the position of
the OH group was critical in determination of scavenging activity.
Compounds with a hydroxyl group at the 5 position of the ring B,
exhibited the most potent scavenging activities (2, 11). After the
compound 2 capturing the metal ions like Cu2þ, Fe3þ and Zn2þ, its
25
26
Resveratrolc
Curcuminc
a
IC50 values were expressed as mean SD for three determinations.
b
Inhibition of A
method was used, the mean SD of at least three independent experiments and the
measurements were carried out in the presence of 20 M compounds.
Resveratrol and curcumin were used as positive control.
b(1e42) self-induced aggregation, the thioflavin-T fluorescence
m
c

N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
543
metal complexes (18, 22, 23) could retain almost the same scav-
enging activities. In compounds 2, 11, 18, 22, 23 especially the
combination of two OH group substitutions at the 2,5 positions of
ring B is correlated with the highest scavenging response. The
simultaneous presence of the two para-position OH groups en-
hances activities. A number of two OH group substitutions at the
2,4 positions of ring B derivatives showed reduced activities (3, 10,
14, 16,17, 21, 24, 25). Replacement of the 5-hydroxyl group on ring B
by a chlorine group was found to reduce the ability of scavenging
the DPPH free radical (5, 8 and 15). This observation indicated that
the chlorine group was negative for the activity compared to the
hydroxyl group. IC50 values of compounds 8e15, 17, 21 and 25
indicated that no matter whether a CO2CH3 group or Cl atom on
ring A was contributed relatively less to the DPPH-scavenging.
Among the target compounds, compounds 2, 11, 18, 22 and 23
compounds exhibited moderate-to-good potencies (25.5e73.1 % at
20
resveratrol (63.7% at 20
aggregation inhibition potency (73.1% at 20
compound 6. Compounds 2, 18, 22, 23 which exhibited the most
potent radical-scavenging activity, had good A aggregation inhi-
bition property (70.3%, 71.8%, 69.6% and 67.5% at 20 M). From the
mM) compared to those of curcumin (56.9% at 20
mM) and
mM). Noticeably, the optimal A
b
(1e42)
m
M) was provided by
b
m
inhibition values of compounds 2e3, 10e11, 16e18 and 21e24, it
appeared that introduction of hydroxy groups into ring B seemed to
be beneficial to A
b self-induced aggregation inhibitory activity.
With the exception of compounds 17 and 21, the reduced activities
were observed for compounds (8, 9, 10, 11) bearing CO2CH3 on the
ring A. According to the inhibition values of the metal compounds
16e26, the different kinds of metal irons did not seem to play a role
since the capturing compound 2 on Cu2þ, Fe3þ and Zn2þ respec-
tively (18, 22 and 23) did not influence dramatically the outcome of
the experiments.
(IC50 ¼ 21.0, 19.0, 17.0, 27.0 and 16.0
mM) showed the most potent
scavenging activity, which was 5.2, 5.7, 6.4, 4.0 and 6.8 times
stronger than that of the reference compound resveratrol
(IC50 ¼ 109.4
mM).
2.5. Docking study of compound 2 with Ab(1e42)
2.3. In vitro antioxidant activity assays
To further study the interaction mode of compound 2 for A
molecular docking study was performed using software package
MOE 2008.10. The X-ray crystal structure of the protein A (1e42)
structure (PDB code 1IYT) [39] used in the docking study was ob-
tained from the Protein Data Bank. As shown in Fig. 4, ring B of
b,
Some compounds tested for their DPPH radical scavenging ac-
tivities were also tested for their antioxidant activities by using the
ABTS (2, 20-azino-bis (3-ethylbenzthiazoline-6-sulfonicacid))
radical scavenging method [37]. Trolox, a water-soluble vitamin E
analog, was used as a reference standard. Their antioxidant activ-
ities were provided as a trolox equivalent, with their relative po-
b
compound 2 interacted with the Ser8 via pep stacking interaction
with the distance of 4.51 Å. A hydrogen bond interaction were
found between ring B of compound 2 and His6, Arg5 residues with
the distance of 4.32 and 3.76 Å, respectively. These results indicated
that the hydrogen bond interactions played important roles in the
tency at 25
mM compared with trolox. As shown in Fig. 2,
compounds 2, 4, 11, 18, 22, 23 had the ability to scavenge the ABTS
radical with 2.6, 1.6, 1.8, 1.7, 0.8, 2.0 trolox equivalents respectively.
All of these selected compounds demonstrated good antioxidant
activities ranging from 0.8- to 2.6-fold of the trolox value. The best
activity was observed for compounds bearing two para-position OH
groups on the ring B. After the compound 2 captured the metal ions
like Cu2þ, Fe3þ and Zn2þ, its metal complexes (18, 22, 23) showed
decreased scavenging activities.
stability of the 2/Ab (1e42) complex.
2.6. Metal chelating effect
The chelating effect of all compounds for metals such as Cu2þ
and Fe2þ in methanol was studied by UVevis spectrometry with
wavelength ranging from 200 to 500 nm [40,41]. In Fig. 5(a),
UVevis spectra of compound 2 at increasing Cu2þ concentrations
were shown as an example. The increase in absorbance, which
could be better estimated by an inspection of the differential
spectra (Fig. 5(b)), indicated that there was an interaction between
Cu2þ and compound 2. Similar behavior was also observed when
using Fe2þ. These observations indicated that our compounds
could effectively chelate Cu2þ and Fe2þ, and thereby could serve as
metal chelators in treating AD. The ratio of ligand/metal ion in the
complex was investigated by mixing the fixed amount metal ion
with increasing ligand; it was possible to observe that the
maximum intensity of difference spectra was reach at about 1:1
ratio, which was taken as an indication of the stoichiometry of the
complex.
2.4. Inhibition of Ab (1e42) self-induced aggregation
All compounds tested for their DPPH radical scavenging activ-
ities were also tested for their ability to inhibit A (1e42) self-
induced aggregation by using a thioflavin-T based fluorometric
assay [38]. Curcumin (Cur) and resveratrol (Res) were used as
reference compounds and the results are summarized in Table 1
and shown in Fig. 3. From the results, it could be seen that most
b
2.7. Inhibition of Cu2þ-induced A
b(1e42) aggregation
To investigate the ability of the salphen derivatives to inhibit
Cu2þ-induced A
(1e42) aggregation, we studied compounds 2, 4
b
and 11 by a ThT-binding assay [42]. Resveratrol and clioquinol were
used as reference compounds. It could be seen from Fig. 6, the
fluorescence of A
which indicates that Cu2þ accelerates A
the fluorescence of A
treated with Cu2þ and the tested compounds
decreased dramatically (2, 85.7% inhibition of Cu2þ-induced A
b
treated with Cu2þ is 146.7% that of A
b
alone,
b
aggregation. By contrast,
b
b
aggregation; 4, 72.4% inhibition; 11, 77.1% inhibition; CQ, 65.7%
inhibition; Res, 60.4% inhibition). These results suggested that our
Fig. 2. The ABTS radical scavenging activities of salphen derivatives and their metal
complexes. Trolox, a water-soluble vitamin E analog, was used as a reference standard
(1.0 equiv).
compounds could inhibit Cu2þ-induced A
b aggregation effectively
by chelating Cu2þ
.

544
N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
Fig. 3. Inhibition of A
b
(1e42) self-induced aggregation by compounds (1e26) comparing with those of curcumin (Cur) and resveratrol (Res). The thioflavin-T fluorescence method
was used and the measurements were carried out in the presence of 20
mM test compound. The mean SD values from three independent experiments were shown.
2.8. Control of Cu2þ-A
b
H2O2 production by compound 2
oxidative stress were assayed using the human neuroblastoma cell
line SH-SY5Y [44]. Compounds 2, 4, 11, 18, 22 and 23 were selected
Binding of redox active metal ions such as Cu2þ to A
known to be involved in generation of ROS such as H2O2 and sub-
b
species is
as representative compounds of different types. First, the colori-
metric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide] assay was performed to examine the potential
cytotoxic effects of compounds 2, 4,11,18, 22 and 23. As indicated in
Fig. 8, compounds 2, 4, 11, 18, 22 and 23 did not show significant
sequent facilitation of Ab aggregation and neurotoxicity. The effect
of compound 2 on H2O2 production by Cu2þeAb42 species was
examined using the HRP/Amplex Red assay [43]. Under reducing
conditions, the Cu2þeA
b
(1e42) react with O2 to generate H2O2.
Addition of compound 2 to such a solution reduced the production
of H2O2 by about 81% for the Cu2þeA
(1e42) species. By com-
effect on cell viability at 1e50
mM after incubation for 24 h. This
suggested that compounds 2, 4 and 11 were nontoxicity to SH-SY5Y
b
cells, and after the compound 2 capturing the metal ions like Cu2þ
,
parison, the strong chelator ethylenediaminetetraacetic acid
(EDTA) showed an even more pronounced effect, almost complete
eliminating (>89%), and clioquinol (CQ) showed the reducing effect
by about 74%. These results showed that compound 2 was able to
chelate metal ions as well as regulated ROS production, which
showed promise for their further applications (Fig. 7).
Fe3þ and Zn2þ, its metal complexes (18, 22, 23) also showed non-
toxicity to SH-SY5Y cells.
Compounds 2, 11, 18 and 23 were tested for their capacity to
protect human SH-SY5Y neuroblastoma cells against oxidative
stress-associated death induced by H2O2. Trolox and resveratrol
were also used as the reference compounds. In this assay, addition
of 200 mM H2O2 to the growth medium reduced cell viability to
65.7% compared to control. The tested compounds were added to
the media at different concentrations immediately prior to the
H2O2 insult. As can be seen in Fig. 9, all of the compounds exhibited
neuroprotective effects at concentrations ranging from 1.25 to
2.9. Cell viability and neuroprotection studies
To gain insight into the therapeutic potential of these de-
rivatives, cell viability and neuroprotective capacity against
Fig. 4. Docking study of compound 2 (colored yellow) with A
compound 2 (colored yellow) and the A (1e42) obtained from docking calculations. The interactions between the ligand and residue Ser8 and His6 are indicated by the green line.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
b (1e42) (PDB code 1IYT). (a) Cartoon representations of compound 2 interacting with Ab (1e42). (b) Association of
b

N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
545
Fig. 6. Inhibition of Cu2þ-induced A
b
(1e42) aggregation by compounds 2, 4 and 11
comparing with those of resveratrol (Res) and clioquinol (CQ) ([A
b
¼
25
m
m
M,
M,
[2] ¼ 50
m
M, [4] ¼ 50
mM, [11] ¼ 50
mM, [Res] ¼ 50
mM, [CQ] ¼ 50
m
M, [Cu2þ] ¼ 25
37 ꢂC, 24 h). Values are reported as the mean SD of three independent experiments.
*p < 0.05, **p < 0.01.
equation and taking into account the limit established by Di et al.
for bloodebrain barrier permeation, we classified compounds as
follows:
(a) ‘CNS þ’ (high BBB permeation predicted); Pe
(10ꢁ6 cm sꢁ1) > 3.5.
(b) ‘CNS
ꢁ’
(low
BBB
permeation
predicted);
Pe
(10ꢁ6 cm sꢁ1) < 1.7.
(c) ‘CNS þ/ꢁ’ (BBB permeation uncertain); Pe (10ꢁ6 cm sꢁ1) from
3.5 to 1.7.
Fig. 5. (a) Uvevis (200e500 nm) absorption spectra of compound 2 (25
mM) in
methanol after addition of ascending amounts of CuCl2 (2e50
mmol/L). (b) The dif-
The Pe values of these selected compounds are summarized in
Table 2. From Table 2, it can be seen that compounds 2, 18, 22 and
23 are able to cross the bloodebrain barrier (BBB).
ferential spectra due to 2-Cu2þ complex formation obtained by numerical subtraction
from the above spectra of those of Cu2þ and 2 at the corresponding concentrations.
10
highest protective capability almost the same as trolox at the
concentration of 10 M and much better than resveratrol. The
metal complex 18 showed moderate protective capabilities similar
to resveratrol at the concentration of 10 M. These observations
mM. Compounds 2, 11 and metal complex 23 had showed the
m
m
further indicated that these new derivatives had the potential to be
efficient multifunctional agents, including antioxidant activity, for
the treatment of AD.
2.10. In vitro bloodebrain barrier permeation assay
In the central nervous system (CNS) drug development, it is
important that the compounds are able to cross the bloodebrain
barrier (BBB). So BBB permeability properties of CNS drug candi-
dates should be determined as early as possible in the drug dis-
covery process. To evaluate the potential for these compounds to
cross the bloodebrain barrier (BBB), we used a parallel artificial
membrane permeation assay for BBB (PAMPA-BBB), which was
described by Di et al. [45,47]. Assay validation was made by
comparing experimental permeability of 9 commercial drugs with
reported values (Table 2). A plot of experimental data versus
Fig. 7. Production of H2O2 from reactions of Ab
, Cu2þ, and compound upon addition of
ascorbate, as determined by an HRP/Amplex-Red assay. Lanes: (1) A
b
; (2) A
b
b
þ Cu2þ
;
(3) A
b
þ Cu2þ þ 2; (4) A
b
þ Cu2þ þ CQ; (5) A
b
þ Cu2þ þ EDTA ([A
] ¼ 200 nM,
[Cu2þ] ¼ 400 nM, [chelator] ¼ 800 nM, [ascorbate] ¼ 10
M, [Amplex Red] ¼ 50 nM,
m
bibliographic values gave
a
good linear correlation, Pe
[HRP] ¼ 0.1 U/mL, and lex/em ¼ 530/590 nm). Values are reported as the mean SD of
(exp.) ¼ 0.8964 Pe (bibl.) e 0.1126 (R2 ¼ 0.9578) (Fig. 10). From this
three independent experiments.

546
N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
Table 2
Permeability (Pe, 10ꢁ6 cm sꢁ1) in the PAMPA-BBB assay for 9 commercial drugs (Used
in the Experiment Validation) and selected salphen derivatives.a
Compounds
Bibl.b Pe (10ꢁ6 cm sꢁ1
)
Compounds Pe (10ꢁ6 cm sꢁ1
)
Testosterone
Clonidine
Hydrocortisone
Corticosterone
Verapamil
17.0
5.3
1.9
15.0
5.2
0.82
3.21
10.08
13.2
6.7
2
4
11
18
22
23
e
5.7
2.9
2.7
9.5
5.7
3.6
e
5.1
16.0
12.0
9.3
b-Estradiol
Progesterone
Piroxicam
2.5
1.6
e
e
Dopamine
0.2
0.3
e
e
a
Experimental data are the mean SD of 3 independent experiments, using PBS:
EtOH (70:30) as solvent.
b
Fig. 8. Effects of compounds on cell viability in SH-SY5Y cells. The cell viability was
determined by the MTT assay after 24 h of incubation with various concentrations of 2,
4, 11, 18, 22 and 23. The results were expressed as a percentage of control cells. Values
are reported as the mean SD of three independent experiments.
Data from Ref. [46].
Qingdao, China) plates and the spots were detected under UV light
(254 nm). Melting point was measured on an XT-4 micromelting
point instrument and uncorrected. IR (KBr-disc) spectra were
recorded by Bruker Tensor 27 spectrometer. 1H NMR and 13C NMR
spectra were measured on a Bruker ACF-500 spectrometer at 25 ꢂC
3. Conclusion
In conclusion, a series of salphen derivatives were designed and
synthesized as multi-functional agents for the treatment of AD.
Among the synthesized compounds, compound 2 exhibited sig-
and referenced to TMS. Chemical shifts were reported in ppm (d)
using the residual solvent line as internal standard. Splitting pat-
terns were designed as s, singlet; d, doublet; t, triplet; m, multiplet.
Mass spectra were obtained on an MS Agilent 1100 Series LC/MSD
Trap mass spectrometer (ESI-MS). Elemental analyses were carried
out on PerkinElmer 2400 Series II CHNS/O System, College of
Pharmacy, China Pharmaceutical University, Nanjing, China.
nificant inhibition of self-induced and Cu2þ-induced A
b
aggrega-
tion, antioxidant activity, metal-chelating ability, control of
Cu2þeA
H2O2 production and low cell toxicity. Furthermore,
compound 2 showed neuroprotective capability much better than
resveratrol at the concentration of 10 M, and it was able to cross
the bloodebrain barrier. In addition, compound 2 retained the ac-
b
m
4.1.1. General procedures for the preparation of compounds 1e26
Two equivalents of the respective salicylaldehyde derivatives
(7.0 mmol) in ethanol (20 mL) were added dropwise to a solution of
one equivalent of 1,2-phenylenediamine derivatives in ethanol
(10 mL). The mixture was stirred under reflux for 1e5 h and then
allowed to cool down to room temperature. The product was
collected, washed with ethanol and dried (P2O5). The obtained
ligand (0.6 mmol) was dissolved in ethanol (10 mL) and heated to
reflux in the presence one equivalent of the respective FeCl3$6H2O,
Cu(OAc)2$H2O and Zn(OAc)2$2H2O in ethanol (5 mL). After 1e6 h,
the mixture was cooled to room temperature, the solid was filtered
off, washed with ethanol and dried in vacuo.
tivities of antioxidant, anti A
b aggregation and neuroprotection
after capturing the metal ions Cu2þ, Fe3þ and Zn2þ (its metal
complexes 18, 22 and 23). These properties highlighted that the
compound 2 could serve as a new multifunctional drug candidate
in the treatment of Alzheimer's disease.
4. Experimental section
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Sino-
pharm Chemical Reagent Co., Ltd. (China). Reaction progress was
monitored using analytical thin layer chromatography (TLC) on
precoated silica gel GF254 (Qingdao Haiyang Chemical Plant,
4.1.1.1. 2,20-((1E,10E)-(1,2-Phenylenebis(azanylylidene))bis(methany-
lylidene))diphenol (1). Yield 60%, light yellow solid, m.p.
Fig. 9. Neuroprotection against H2O2 toxicity. Compounds 2, 11, 18 and 23 were tested for neuroprotective activity against H2O2 toxicity in SH-SY5Y neuroblastoma cell cultures.
Resveratrol (10 M) was used as the reference compound. Results are expressed as percent viability compared to cells not treated with H2O2. Data represent the mean SD of three
observations. ***p < 0.001, *p < 0.05, **p < 0.01.
m

N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
547
(s, 2H), 7.79 (d, J ¼ 2.7 Hz, 2H), 7.51e7.43 (m, 6H), 7.02 (d, J ¼ 8.8 Hz,
2H). 13C NMR (125 MHz, DMSO)
162.62, 159.44, 142.45, 133.26,
d
131.14, 128.47, 122.93, 121.24, 120.04, 119.12. ESI-MS m/z: 383.0
[MꢁH]ꢁ.
4.1.1.6. (N1E,N2E)-N1,N2-Bis(pyridin-2-ylmethylene)benzene-1,2-
diamine (6). Yield 63%, yellow solid, m.p. 212e213 ꢂC, IR (KBr)
n
1646.84, 1583.64, 1464.84, 1438.48, 1388.72, 1312.21, 1209.00,
1151.13, 1114.34, 777.97, 724.04 cmꢁ1 1H NMR (500 MHz, CDCl3)
.
d
8.96 (d, J ¼ 8.5 Hz, 1H), 8.43 (d, J ¼ 5.9 Hz, 1H), 8.39 (d, J ¼ 4.8 Hz,
1H), 7.73e7.64 (m, 2H), 7.48 (d, J ¼ 7.6 Hz,1H), 7.41 (t, J ¼ 7.4 Hz,1H),
7.12e7.02 (m, 2H), 7.02e6.96 (m, 2H), 6.47 (d, J ¼ 8.0 Hz, 1H), 5.22
(s, 2H). 13C NMR (125 MHz, CDCl3)
d 162.04, 154.75, 153.07, 148.75,
147.57, 141.28, 138.23, 137.59, 131.18, 131.11, 127.40, 126.00, 123.23,
118.55, 118.35, 116.59, 115.21. ESI-MS m/z: 287.0 [MþH]þ.
4.1.1.7. 1,10-((1E,10E)-(1,2-Phenylenebis(azanylylidene))bis(methany-
lylidene))bis (naphthalen-2-ol) (7). Yield 77%, yellow solid, m.p.
Fig. 10. Linear correlation between experimental and reported permeability of com-
mercial drugs using the PAMPA-BBB assay.
213 ꢂC, IR (KBr)
n
1619.96, 1589.59, 1472.84, 1399.63, 1326.60,
1251.65, 1176.42, 968.39, 884.26, 820.45, 736.91 cmꢁ1 1H NMR
(500 MHz, DMSO)
.
167e168 ꢂC, IR (KBr)
1276.49, 1192.09, 1150.28, 1114.95, 909.96, 855.03, 830.60, 810.11,
787.25, 760.44, 639.92, 581.40, 510.45 cmꢁ1 1H NMR (500 MHz,
n 1614.02, 1561.94, 1480.45, 140.15, 1361.88,
d
15.10 (d, J ¼ 3.6 Hz, 2H), 9.69 (d, J ¼ 3.5 Hz, 2H),
8.54 (d, J ¼ 8.5 Hz, 2H), 7.96 (d, J ¼ 9.2 Hz, 2H), 7.82 (d, J ¼ 9.0 Hz,
4H), 7.55 (t, J ¼ 7.6 Hz, 2H), 7.44 (dd, J ¼ 6.0, 3.4 Hz, 2H), 7.37 (t,
J ¼ 7.4 Hz, 2H), 7.07 (d, J ¼ 9.1 Hz, 2H). 13C NMR (125 MHz, DMSO)
.
DMSO)
d 12.96 (s, 2H), 8.95 (s, 2H), 7.71e7.67 (m, 2H), 7.48 (dd,
J ¼ 5.9, 3.5 Hz, 2H), 7.46e7.40 (m, 4H), 6.99 (t, J ¼ 7.3 Hz, 4H). 13
C
d
168.81,157.83,139.03,137.09,133.41,129.35,128.51,127.75,127.30,
123.98, 121.81, 121.00, 120.13, 109.68. ESI-MS m/z: 417.0 [MþH]þ.
NMR (125 MHz, DMSO)
d 164.51, 160.89, 142.76, 133.90, 132.94,
128.26, 120.24, 119.99, 119.55, 117.16. ESI-MS m/z: 315.0 [MꢁH]ꢁ.
4.1.1.8. Methyl 3,4-bis((E)-(5-chloro-2-hydroxybenzylidene)amino)
4.1.1.2. 2,20-((1E,10E)-(1,2-Phenylenebis(azanylylidene))bis(methany-
benzoate (8). Yield 52%, light yellow solid, m.p.192e194 ꢂC, IR (KBr)
lylidene))bis (benzene-1,4-diol) (2). Yield 64%, red solid, m.p.
n
1717.50, 1618.36, 1562.63, 1477.18, 1434.95, 1356.21, 1307.72,
1279.16, 1182.21, 1120.24, 1190.11, 819.84, 762.84, 730.40, 649.06,
509.91 cmꢁ1 1H NMR (500 MHz, DMSO)
12.75 (s, 1H), 12.46 (s,
226e227 ꢂC, IR (KBr)
n
1619.64, 1572.10, 1483.32, 1352.75, 1299.69,
1209.68, 1156.74, 1047.41, 989.12, 885.24, 786.97, 747.04 cmꢁ1 1H
NMR (500 MHz, DMSO) 12.14 (s, 2H), 9.09 (s, 2H), 8.80 (s, 2H), 7.41
.
.
d
d
1H), 9.00 (s,1H), 8.93 (s,1H), 7.99 (d, J ¼ 1.7 Hz,1H), 7.96 (dd, J ¼ 8.2,
1.8 Hz, 1H), 7.81 (d, J ¼ 2.7 Hz, 1H), 7.80 (d, J ¼ 2.7 Hz, 1H), 7.52 (d,
J ¼ 8.3 Hz, 1H), 7.45 (ddd, J ¼ 10.3, 8.9, 2.7 Hz, 2H), 7.00 (t, J ¼ 9.1 Hz,
(dd, J ¼ 6.0, 3.4 Hz, 2H), 7.37 (dd, J ¼ 5.8, 3.5 Hz, 2H), 7.04 (d,
J ¼ 2.9 Hz, 2H), 6.87 (dd, J ¼ 8.8, 2.9 Hz, 2H), 6.79 (d, J ¼ 8.8 Hz, 2H).
13C NMR (125 MHz, DMSO)
d 164.16, 153.65, 149.98, 142.95, 127.92,
2H), 3.90 (s, 3H). 13C NMR (125 MHz, DMSO)
d 165.94, 163.38,
121.71, 120.24, 119.85, 117.61, 117.30. ESI-MS m/z: 383.0 [MꢁH]ꢁ.
163.23, 159.45, 159.39, 146.98, 142.37, 133.77, 133.48, 131.21, 130.80,
129.06, 123.17, 123.02, 121.42, 121.28, 120.65, 120.58, 119.22, 119.14,
52.69. ESI-MS m/z: 441.0 [MꢁH]ꢁ.
4.1.1.3. 4,40-((1E,10E)-(1,2-Phenylenebis(azanylylidene))bis(methany-
lylidene))bis (benzene-1,3-diol) (3). Yield 71%, yellow solid, m.p.
>250 ꢂC, IR (KBr)
n
1611.46, 1576.70, 1501.78, 1357.02, 1307.97,
1233.54, 1209.09, 1183.33, 1153.96, 1123.85, 847.27, 745.15,
532.22 cmꢁ1 1H NMR (500 MHz, DMSO)
13.39 (s, 2H), 10.27 (s,
4.1.1.9. Methyl
amino)benzoate (9). Yield 75%, yellow solid, m.p. 119e200 ꢂC, IR
(KBr) 1711.33, 1613.34, 1467.53, 1440.68, 1290.83, 1253.83,
1093.20, 971.74, 857.63, 769.10, 735.43 cmꢁ1 1H NMR (500 MHz,
DMSO) 12.75 (s, 1H), 12.54 (s, 1H), 9.01 (s, 1H), 8.95 (s, 1H),
3,4-bis((E)-(2-hydroxy-3-methoxybenzylidene)
d
n
2H), 8.74 (s, 2H), 7.43 (d, J ¼ 8.5 Hz, 2H), 7.37 (dt, J ¼ 7.1, 3.6 Hz, 2H),
.
7.34e7.29 (m, 2H), 6.40 (dd, J ¼ 8.5, 2.3 Hz, 2H), 6.30 (d, J ¼ 2.2 Hz,
d
2H). 13C NMR (125 MHz, DMSO)
d 163.72, 163.26, 163.04, 142.44,
7.98e7.94 (m, 2H), 7.54 (d, J ¼ 8.2 Hz, 1H), 7.30 (dd, J ¼ 13.2, 7.3 Hz,
134.77, 127.37, 119.89, 112.70, 108.23, 102.83. ESI-MS m/z: 347.0
2H), 7.15 (t, J ¼ 8.1 Hz, 2H), 6.92 (td, J ¼ 7.9, 4.1 Hz, 2H), 3.90 (s, 3H),
[MꢁH]ꢁ.
3.82 (s, 3H), 3.81 (s, 3H). 13C NMR (125 MHz, DMSO)
d 166.00,
165.58, 165.34, 151.11, 151.03, 148.37, 148.31, 146.86, 142.60, 128.86,
128.76, 124.22, 124.03, 120.77, 119.88, 119.86, 119.14, 119.02, 116.39,
116.22, 56.19, 56.12, 52.64. ESI-MS m/z: 435.0 [MþH]þ.
4.1.1.4. 6,60-((1E,10E)-(1,2-Phenylenebis(azanylylidene))bis(methany-
lylidene))bis (2-methoxyphenol) (4). Yield 55%, red solid, m.p.
172 ꢂC, IR (KBr)
n
1612.74, 1573.95, 1467.09, 1400.89, 1256.55,
1205.60, 1077.70, 972.29, 781.06, 736.66 cmꢁ1. 1H NMR (500 MHz,
DMSO)
4.1.1.10. Methyl 3,4-bis((E)-(2,4-dihydroxybenzylidene)amino)ben-
zoate (10). Yield 54%, light yellow solid, m.p. 233e234 ꢂC, IR (KBr)
n
d
12.99 (s, 2H), 8.91 (s, 2H), 7.45 (dt, J ¼ 7.1, 3.6 Hz, 2H),
7.42e7.37 (m, 2H), 7.25 (dd, J ¼ 7.9, 1.4 Hz, 2H), 7.12 (dd, J ¼ 8.0,
1716.44, 1625.55, 1584.17, 1490.33, 1436.50, 1399.19, 1716.44,
1625.55, 1584.17, 1490.38, 1436.50, 1399.19, 1283.58, 1266.09,
1.2 Hz, 2H), 6.90 (t, J ¼ 7.9 Hz, 2H), 3.81 (s, 6H). 13C NMR (125 MHz,
1216.45, 1127.61, 992.15, 877.03, 787.03, 763.45 cmꢁ1 1H NMR
.
DMSO)
d 164.63, 151.08, 148.31, 142.50, 128.14, 124.19, 120.19,
119.79, 118.91, 115.98, 56.14. ESI-MS m/z: 375.0 [MꢁH]ꢁ.
(500 MHz, DMSO)
d
11.92 (s, 1H), 11.73 (s, 1H), 9.11 (d, J ¼ 12.6 Hz,
2H), 8.89 (s, 1H), 8.84 (s, 1H), 7.93 (d, J ¼ 5.9 Hz, 2H), 7.49 (d,
J ¼ 8.7 Hz, 1H), 7.08 (dd, J ¼ 8.3, 2.9 Hz, 2H), 6.89 (td, J ¼ 8.5, 3.0 Hz,
2H), 6.79 (t, J ¼ 8.2 Hz, 2H), 3.90 (s, 3H). 13C NMR (125 MHz, DMSO)
4.1.1.5. 2,20-((1E,10E)-(1,2-Phenylenebis(azanylylidene))bis(methany-
lylidene))bis (4-chlorophenol) (5). Yield 67%, red solid, m.p.
213e214 ꢂC, IR (KBr)
n
1614.36, 1565.17, 1475.69, 1384.97, 1350.11,
1273.84, 1189.43, 873.28, 845.21, 816.83, 790.39, 755.38, 716.03,
646.25, 499.33 cmꢁ1. 1H NMR (500 MHz, DMSO)
12.90 (s, 2H), 8.94
d 166.07, 164.98, 164.76, 153.70, 153.65, 150.09, 150.91, 147.35,
143.02,128.62,128.52,122.35,122.02,120.82,120.78,119.94,119.90,
d
117.75, 117.64, 117.20, 116.93, 52.61. ESI-MS m/z: 405.0 [MꢁH]ꢁ.

548
N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
4.1.1.11. Methyl 3,4-bis((E)-(2,5-dihydroxybenzylidene)amino)benzo-
ate (11). Yield 55%, yellow solid, m.p. >250 ꢂC, IR (KBr)
1709.43,
1615.58, 1584.84, 1515.28, 1350.58, 1334.17, 1304.80, 1243.28,
1215.39, 1116.95, 986.29, 845.03, 766.39, 534.86 cmꢁ1 1H NMR
(500 MHz, DMSO)
4 . 1 . 1 . 1 7 . N , N 0- B i s - 4 - ( h y d r o x y s a l i c y l i d e n e ) 4 -
n
methylesterphenylenediamineecopper(II) (17). Yield 63%, dark yel-
low solid, m.p. >250 ꢂC; IR (KBr)
n
3451.21,1697.04,1619.07,1531.49,
1442.61, 1370.86, 1336.01, 1238.66, 1194.26, 1139.01, 983.90, 842.88,
798.44, 762.01, 658.07, 636.64, 573.85, 538.85, 501.57, 474.88 cmꢁ1
.
d
13.11 (d, J ¼ 12.3 Hz, 2H), 10.32 (s, 2H), 8.83 (s,
.
1H), 8.80 (s, 1H), 7.91e7.86 (m, 2H), 7.52e7.45 (m, 3H), 6.41 (ddd,
Anal. Calcd for C22H16CuN2O6: C, 56.47; H, 3.45; N, 5.99. Found: C,
J ¼ 8.5, 4.0, 2.3 Hz, 2H), 6.30 (t, J ¼ 2.4 Hz, 2H), 3.88 (s, 3H). 13C NMR
56.58; H, 3.51; N, 5.94. ESI-MS m/z: 466.0 [MꢁH]ꢁ.
(125 MHz, DMSO)
d 166.13, 164.20, 163.98, 163.91, 163.66, 163.62,
163.32, 146.73, 142.67, 134.98, 134.94, 128.12, 128.06, 120.52, 120.32,
112.72, 108.61, 108.40, 102.86, 102.81, 52.55. ESI-MS m/z: 407.0
[MþH]þ.
4 .1.1.18 . N , N 0- B i s - 5 - ( h y d r o x y s a l i c y l i d e n e ) p h e n y l -
enediamineecopper(II) (18). Yield 56%, brown solid, m.p. >250 ꢂC;
IR (KBr)
n 3444.20, 1608.22, 1536.64, 1470.80, 1385.34, 1317.80,
1209.88, 1158.57, 967.05, 824.54, 748.44, 493.66, 438.02 cmꢁ1. Anal.
Calcd for C20H14CuN2O4: C, 58.61; H, 3.44; N, 6.83. Found: C, 58.51;
H, 3.48; N, 6.81. ESI-MS m/z: 408.0 [MꢁH]ꢁ.
4.1.1.12. 1,10-((1E,10E)-((4-Chloro-1,2-phenylene)bis(azanylylidene))
bis (methanylylidene))bis(naphthalen-2-ol) (12). Yield 69%, yellow
solid, m.p. 254e255 ꢂC, IR (KBr)
n
1617.88, 1565.37, 1481.42, 1399.61,
1351.43, 1308.89, 1215.74, 1180.77, 969.46, 908.13, 818.54, 735.92,
470.94, 417.95 cmꢁ1 1H NMR (500 MHz, DMSO)
14.95 (d,
4.1.1.19. N, N0-Bis-2-(hydroxynaphthaldehyde)phenyl-
enediamineecopper(II) (19). Yield 77%, yellow solid, m.p. >250 ꢂC;
.
d
J ¼ 13.7 Hz, 2H), 9.70 (d, J ¼ 13.0 Hz, 2H), 8.58 (dd, J ¼ 10.1, 8.5 Hz,
2H), 8.07e7.92 (m, 3H), 7.83 (d, J ¼ 8.4 Hz, 3H), 7.55 (t, J ¼ 7.0 Hz,
2H), 7.46 (d, J ¼ 10.5 Hz,1H), 7.38 (t, J ¼ 7.4 Hz, 2H), 7.07 (dd, J ¼ 12.1,
IR (KBr) n 3452.38, 2359.21, 1611.63, 1534.16, 1459.78, 1399.06,
1366.31, 1192.28, 831.15, 736.92, 655.57, 559.90, 495.59,
475.00 cmꢁ1. Anal. Calcd for C28H18CuN2O2: C, 70.36; H, 3.80; N,
5.86. Found: C, 70.31; H, 3.84; N, 5.75. ESI-MS m/z: 478.0 [MþH]þ.
9.2 Hz, 2H). 13C NMR (125 MHz, DMSO)
d 168.85, 168.04, 158.60,
158.58, 140.33, 138.44, 137.43, 137.12, 133.40, 133.30, 131.88, 129.36,
129.34, 128.53, 127.41, 127.38, 127.13, 124.13, 124.08, 121.69, 121.46,
121.36, 121.15, 119.82, 109.86, 109.83. ESI-MS m/z: 451.0 [MþH]þ.
4.1.1.20. N,N0-Bis(salicylidene)phenylenediamineeiron(Ⅲ) chloride
(20). Yield 45%, brown solid, m.p. >250 ꢂC; IR (KBr)
n 3451.01,
1604.24, 1533.91, 1462.69, 1439.51, 1380.25, 1315.91, 1196.89,
1149.86, 1127.55, 923.77, 872.57, 812.96, 760.53, 615.16, 539.42,
493.12, 476.01, 428.77 cmꢁ1. Anal. Calcd for C20H14ClFeN2O2: C,
59.22; H, 3.48; N, 6.91. Found: C, 59.35; H, 3.31; N, 6.82. ESI-MS m/z:
406.0 [MþH]þ.
4.1.1.13. 6,60-((1E,10E)-((4-Chloro-1,2-phenylene)bis(azanylylidene))
bis (methanylylidene))bis(2-methoxyphenol) (13). Yield 77%, yellow
solid, m.p. 215 ꢂC, IR (KBr)
n
1614.07, 1464.74, 1408.09, 1253.03,
1207.52, 971.74, 780.18, 737.27 cmꢁ1 1H NMR (500 MHz, DMSO)
12.71 (d, J ¼ 12.1 Hz, 2H), 8.96 (s, 1H), 8.92 (s, 1H), 7.59 (d,
.
d
J ¼ 1.8 Hz, 1H), 7.46 (dt, J ¼ 8.5, 5.2 Hz, 2H), 7.29e7.23 (m, 2H), 7.14
4 . 1 . 1 . 2 1 . N , N 0- B i s - 4 - ( h y d r o x y s a l i c y l i d e n e ) 4 -
(dd, J ¼ 7.4, 4.1 Hz, 2H), 6.91 (td, J ¼ 7.9, 3.9 Hz, 2H), 3.81 (s, 6H). 13
C
methylesterphenylenediamineeiron(III) chloride (21). Yield 64%,
NMR (125 MHz, DMSO)
d
165.63, 164.93, 151.03, 150.96, 148.31,
brown solid, m.p. >250 ꢂC; IR (KBr)
n 3388.82, 1706.19, 1611.62,
143.76, 141.60, 132.03, 127.58, 124.24, 124.10, 121.72, 120.06, 119.81,
119.77, 119.05, 119.02, 116.27, 116.14, 56.16. ESI-MS m/z: 409.0
[MꢁH]ꢁ.
1579.40, 1538.80, 1439.67, 1378.25, 1242.90, 1208.59, 1136.27,
986.34, 853.14, 804.05, 766.01, 651.58, 635.19, 604.43, 533.06,
507.19, 463.15 cmꢁ1. Anal. Calcd for C22H16ClFeN2O6: C, 53.31; H,
3.25; N, 5.65. Found: C, 53.42; H, 3.24; N, 5.66. ESI-MS m/z: 494.0
[MꢁH]ꢁ.
4.1.1.14. 4,40-((1E,10E)-((4-Chloro-1,2-phenylene)bis(azanylylidene))
bis (methanylylidene))bis(benzene-1,3-diol) (14). Yield 66%, yellow
solid, m.p. >250 ꢂC, IR (KBr)
n
1613.60, 1540.26, 1500.05, 1349.45,
1333.66, 1260.00, 1209.41, 1161.93, 1131.19, 982.09, 850.65, 805.52,
683.00, 520.33 cmꢁ1. 1H NMR (500 MHz, Acetone)
13.21 (s, 1H),
4 .1.1. 2 2 . N , N 0- B i s - 5 - ( h y d r o x y s a l i c y l i d e n e ) p h e n y l -
enediamineeiron(III)chloride (22). Yield 71%, black solid, m.p.
d
>250 ꢂC; IR (KBr)
n
3443.76, 1603.35, 1538.08, 1466.45, 1305.14,
1226.99, 1205.09, 1159.46, 967.91, 822.15, 754.32, 729.12, 687.88,
599.98, 539.51, 506.64, 476.06 cmꢁ1
Anal. Calcd for
13.12 (s, 1H), 9.19 (s, 2H), 8.77 (s, 1H), 8.73 (s, 1H), 7.41 (m, 4H), 7.33
(dd, J ¼ 8.5, 2.2 Hz, 1H), 6.47 (ddd, J ¼ 8.6, 6.7, 2.2 Hz, 2H), 6.38 (s,
.
2H). 13C NMR (125 MHz, Acetone)
d
165.40, 164.84, 164.43, 164.37,
C20H14ClFeN2O4: C, 54.89; H, 3.22; N, 6.40. Found: C, 54.80; H, 3.26;
163.83, 163.66, 135.87, 135.65, 132.46, 127.43, 122.00, 120.59, 113.71,
N, 6.56. ESI-MS m/z: 436.0 [MꢁH]ꢁ.
113.66, 108.79, 108.70, 103.47. ESI-MS m/z: 383.0 [MþH]þ.
4.1.1.23. N,N0-Bis-5-(hydroxysalicylidene)phenylenediamineezinc(II)
(23). Yield 55%, yellow solid, m.p. >250 ꢂC; IR (KBr)
n 3355.24,
4.1.1.15. 2,20-((1E,10E)-((4-Chloro-1,2-phenylene)bis(azanylylidene))
bis (methanylylidene))bis(4-chlorophenol) (15). Yield 59%, yellow
1616.69, 1542.89, 1476.86, 1441.75, 1393.20, 1294.48, 1244.24,
1219.74, 1157.53, 965.83, 876.03, 858.26, 827.57, 806.73, 749.36,
solid, m.p. 208e209 ꢂC, IR (KBr)
n
1617.02, 1558.27, 1476.47, 1353.56,
1276.47, 1184.62, 1119.08, 1090.25, 930.58, 857.06, 836.75, 743.09,
658.81, 506.30 cmꢁ1 1H NMR (500 MHz, DMSO)
12.63 (d, 2H),
599.09, 553.03, 486.90 cmꢁ1. 1H NMR (500 MHz, DMSO)
d 8.89 (s,
.
d
2H), 8.49 (s, 2H), 7.88 (dd, J ¼ 6.1, 3.5 Hz, 2H), 7.36 (dd, J ¼ 6.1, 3.4 Hz,
8.94 (s, 1H), 8.91 (s, 1H), 7.76 (s, 2H), 7.58 (d, J ¼ 1.2 Hz, 1H),
2H), 6.86 (dd, J ¼ 9.0, 3.2 Hz, 2H), 6.79 (d, J ¼ 3.2 Hz, 2H), 6.61 (d,
7.51e7.41 (m, 4H), 7.00 (dd, J ¼ 8.8, 3.5 Hz, 2H). 13C NMR (125 MHz,
J ¼ 9.0 Hz, 2H). 13C NMR (125 MHz, DMSO)
d 167.38, 162.31, 145.15,
139.88, 127.45, 125.64, 123.94, 118.59, 118.43, 116.80. ESI-MS m/z:
DMSO)
d
163.48, 162.74, 159.37, 159.34, 143.75, 141.37, 133.56,
133.39, 132.35, 131.07, 130.99, 127.87, 123.05, 123.02, 121.50, 121.25,
411.0 [MþH]þ.
121.21, 119.94, 119.14, 119.11. ESI-MS m/z: 419.0 [MþH]þ.
4.1.1.24. N,N0-Bis-4-(hydroxysalicylidene)phenylenediamineezinc(II)
(24). Yield 59%, yellow solid, m.p. >250 ꢂC, IR (KBr)
n 3424.10,
4 .1.1.16 . N , N 0- B i s - 4 - ( h y d r o x y s a l i c y l i d e n e ) p h e n y l -
enediamineecopper(II) (16). Yield 74%, yellow solid, m.p. >250 ꢂC;
1613.75, 1560.91, 1479.28, 1449.12, 1394.17, 1356.97, 1239.00,
1189.54, 1132.87, 990.86, 844.05, 792.41, 748.24, 653.87, 526.55,
IR (KBr)
n 3442.44, 2921.08, 1605.96, 1555.87, 1452.04, 1369.86,
1237.45, 1206.12, 1117.78, 849.99, 792.24, 762.01, 646.76, 533.85,
495.30 cmꢁ1. Anal. Calcd for C20H14CuN2O4: C, 58.61; H, 3.44; N,
6.83. Found: C, 58.57; H, 3.39; N, 6.86. ESI-MS m/z: 408.0 [MꢁH]ꢁ.
487.22 cmꢁ1. 1H NMR (500 MHz, DMSO)
d 9.75 (s, 2H), 8.82 (s, 2H),
7.78 (dd, J ¼ 6.1, 3.5 Hz, 2H), 7.28 (dd, J ¼ 6.1, 3.4 Hz, 2H), 7.23 (d,
J ¼ 9.1 Hz, 2H), 6.10e6.06 (m, 4H). 13C NMR (125 MHz, DMSO)

N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
549
d
174.78, 164.13, 161.03, 139.89, 138.35, 126.50, 116.05, 114.30,
to four times using plastic pipettes. The optical absorbance of ABTS
at 415 nm was measured at 6 min after addition and equilibrated at
30 ꢂC. Each individual treatment was repeated for three times and
the results of the experiments were compared.
106.93, 105.15. ESI-MS m/z: 411.0 [MþH]þ.
4 . 1 . 1 . 2 5 . N , N 0- B i s - 4 - ( h y d r o x y s a l i c y l i d e n e ) - 4 -
chlorophenylenediamineezinc (II) (25). Yield 79%, yellow solid, m.p.
>250 ꢂC, IR (KBr)
n
3407.85, 1619.26, 1547.46, 1476.18, 1450.11,
1389.76, 1362.30, 1220.00, 1178.54, 1130.47, 926.24, 843.57, 643.92,
535.81, 510.45, 420.76 cmꢁ1. 1H NMR (500 MHz, DMSO)
9.83 (s,
4.4. Inhibition of Ab (1e42) self-induced aggregation
d
Inhibition of A
oflavin T (ThT)-binding assay [38]. HFIP pretreated A
samples (Anaspec Inc) were resolubilized with a 50 mM phosphate
buffer (pH 7.4) to give a 25 M solution. Each tested compound was
firstly prepared in DMSO at a concentration of 10 mM and 1 L of
b
(1e42) aggregation was measured using a Thi-
2H), 8.86 (s, 1H), 8.82 (s, 1H), 7.90 (d, J ¼ 1.6 Hz, 1H), 7.80 (d,
b
(1e42)
J ¼ 8.8 Hz, 1H), 7.32e7.20 (m, 3H), 6.09 (d, J ¼ 8.7 Hz, 4H). 13C NMR
(125 MHz, DMSO)
d
175.20, 174.96, 164.57, 164.35, 161.93, 161.28,
m
141.15, 138.80, 138.63, 138.42, 130.87, 125.83, 117.68, 116.16, 114.30,
m
114.24, 106.88, 106.85, 105.54, 105.36. ESI-MS m/z: 445.0 [MþH]þ.
each was added to the well of black, opaque Corning 96-well plates
such that the final solvent concentration was 10%. The final con-
4.1.1. 26. N, N0-Bis-2-(hydroxynaphthaldehyde)phenyl-
enediamineezinc (II) (26). Yield 66%, red solid, m.p. >250 ꢂC, IR
centration of each compound was 20
dependent triplicates. The solvent control was also included. Then,
L of 25 M A (1e42) sample was added to each well and the
mM and was prepared in in-
(KBr)
1431.24, 1399.16, 1362.66, 1185.44, 979.54, 827.60, 744.41,
661.71 cmꢁ1 1H NMR (500 MHz, DMSO)
9.83 (s, 2H), 8.46 (d,
n
3450.17, 2346.92, 1612.30, 1577.48, 1534.96, 1459.81,
9
m
m
b
samples mixed by gentle trapping. Plates were covered to minimize
evaporation and incubated in dark at room temperature for
.
d
J ¼ 8.5 Hz, 2H), 8.15 (dd, J ¼ 6.1, 3.5 Hz, 2H), 7.82 (d, J ¼ 9.2 Hz, 2H),
7.72 (d, J ¼ 7.8 Hz, 2H), 7.53e7.47 (m, 2H), 7.41 (dd, J ¼ 6.1, 3.3 Hz,
2H), 7.26 (t, J ¼ 7.4 Hz, 2H), 7.02 (d, J ¼ 9.2 Hz, 2H). 13C NMR
46e48 h with no agitation. After the incubation period, 200
mL of
5
mM ThT in 50 mM glycine-NaOH buffer (pH 8.0) was added to
each well. Fluorescence was measured on a SpectraMax M5 (Mo-
lecular Devices, Sunnyvale, CA, USA) multi-mode plate reader with
excitation and emission wavelengths at 446 nm and 490 nm,
respectively. The fluorescence intensities were compared and the
percent inhibition due to the presence of the inhibitor was calcu-
lated by the following formula: 100 ꢁ (IFi/IFo ꢃ 100) where IFi and
(125 MHz, DMSO)
d 174.05, 156.49, 140.62, 136.18, 136.00, 129.17,
127.88, 127.41, 127.07, 126.08, 122.10, 120.01, 117.13, 109.47. ESI-MS
m/z: 479.0 [MþH]þ.
4.2. DPPH free radical-scavenging assay
IFo are the fluorescence intensities obtained for Ab (1e42) in the
DPPH was used to assess free radical-scavenging activity [36].
DPPH is one of the few stable and commercially available organic
nitrogen radicals and has a UVevis absorption maximum at 517 nm.
Upon reduction, the solution color fades; the reaction progress is
conveniently monitored by a spectrophotometer. To test free
radical-scavenging effects, compounds 1e26 were adjusted with
presence and in the absence of inhibitor, respectively.
4.5. Docking study
Molecular modeling calculations and docking studies were
performed using Molecular Operating Environment (MOE) soft-
ware version 2008.10 (Chemical Computing Group, Montreal,
methanol solution to final concentrations of 0e200
DPPH (400 M) was used in the reaction mixture. Serial dilutions of
the test sample (20 L) were combined with the DPPH (180 L,
400 M) solution in a 96-well microtitre plate. MeOH was used as a
mM. Methanolic
m
Canada). The X-ray crystal structure of Ab (1e42) (PDB 1IYT) used in
m
m
the docking study was obtained from the Protein Data Bank (www.
rcsb.org). Heteroatoms and water molecules in the PDB file were
removed at the beginning, and all hydrogen atoms were added to
the protein. Amber99 force field was assigned to the enzyme and
the partial charges were calculated with the same force field. Pro-
tonate states of the enzyme at pH 7 were obtained by following the
Protonate 3D protocol in which all configurations were set as
default. Compound 2 was drawn in MOE with all hydrogen atoms
added. During the docking procedure, pose of compound 2 was
initially generated by Triangle Matcher method, and scored with
london dG function. 30 Poses of the compound were dedicated to
the next refinement procedure. All poses were fine tune with the
force field refinement scheme. The best 10 poses of molecules were
retained and scored. After docking, the geometry of resulting
complex was studied using the MOE's pose viewer utility.
m
negative control and resveratrol was used as positive control. The
reaction mixtures were incubated for 30 min at 37 ꢂC in the dark
and the change in absorbance at 517 nm was measured. Mean
values were obtained from triplicate experiments. Inhibition
percent was calculated using the equation: DPPH radical-
scavenging rate (%) ¼ [1 ꢁ (A ꢁ C)/B] ꢃ 100, where A is the absor-
bance of the sample (DPPH þ compounds), B is the absorbance of
the DPPH radical-methanol solution, and C is the absorbance of the
sample (compounds) alone. Percent inhibition was plotted against
concentration, and the equation for the line was used to obtain the
IC50 value. The IC50 values of samples were compared against the
standard, resveratrol, and the lower the IC50 of synthesized com-
pounds, the better it is as an antioxidant.
4.3. ABTS radical cation scavenging activity assay [46]
4.6. Spectrophotometric measurement of complex with Cu2þ and
Fe2þ
2, 20-Azino-bis-2-ethybenz-thiazoline-6-sulfonic acid (ABTS)
was dissolved in purified water to a 7 mM concentration. ABTS
radical cation (ABTSþ) was produced by reacting ABTS stock solution
with 2.45 mM potassium persulfate (final concentration) and
allowing the mixture to stand in the dark at room temperature for at
least 18 h before use. The stock solution of ABTS was serially diluted
The study of metal chelation was performed in methanol at
298 K using UVevis spectrophotometer (SHIMADZU UV-2450PC)
with wavelength ranging from 200 to 500 nm [40,41]. The differ-
ence UVevis spectra due to complex formation was obtained by
numerical subtraction of the spectra of the metal alone and the
compound alone (at the same concentration used in the mixture)
with sodium phosphate buffer (50 mM, pH 7.4) to 100
4,11,18, 22 and 23 at different concentrations (total volume of 50
were added to 150 L of 100 M ABTS solution, respectively. After
mM. Trolox, 2,
mL)
from the spectra of the mixture. A fixed amount of 2 (25
mmol/L)
m
m
was mixed with growing amounts of copper ion (2e50 mol/L) and
m
the addition of either trolox or another antioxidant to the ABTS so-
lution, complete mixing of reactants was achieved by bubbling three
tested the difference UVevis spectra to investigate the ratio of
ligand/metal in the complex.

550
N. Jiang et al. / European Journal of Medicinal Chemistry 87 (2014) 540e551
4.7. Inhibition of Cu2þ-induced A
b
(1e42)aggregation
pore size 0.45 mm) and the acceptor microplate were both from
Millipore. The 96-well UV plate (COSTAR@) was from Corning
For the inhibition of Cu2þ-induced A
experiment [42], the A was diluted in 20 M HEPES (pH 6.6) with
150 M NaCl. The mixture of the peptide (10 L, 25 M, final con-
centration) with or without copper (10 L, 25 M, final concen-
tration) and the test compound (10 L, 50 M, final concentration)
was incubated at 37 ꢂC for 24 h. The 20
L of the sample was diluted
to a final volume of 200 L with 50 mM glycine-NaOH buffer (pH
b
(1e42) aggregation
Incorporated. The acceptor 96-well microplate was filled with
b
m
300
nated with 4
dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH
(7:3) to achieve a concentration of 100 mg/mL, 200 L of which was
mL of PBS/EtOH (7:3), and the filter membrane was impreg-
m
m
m
mL of PBL in dodecane (20 mg/mL). Compounds were
m
m
m
m
m
m
added to the donor wells. The acceptor filter plate was carefully
placed on the donor plate to form a sandwich, which was left un-
disturbed for 16 h at 25 ꢂC. After incubation, the donor plate was
carefully removed and the concentration of compound in the
acceptor wells was determined using a UV plate reader (Flexsta-
tion@ 3). Every sample was analyzed at five wavelengths, in four
wells, in at least three independent runs, and the results are given
as the mean standard deviation. In each experiment, 9 quality
control standards of known BBB permeability were included to
validate the analysis set.
m
8.0) containing thioflavin T (5
mM). The detection method was the
same as that of self-induced A
b
aggregation experiment.
4.8. Hydrogen peroxide assays
Hydrogen peroxide production was determined using an HRP/
Amplex Red assay. A general protocol from Invitrogen's Amplex Red
Hydrogen Peroxide/Peroxidase Assay as followed [43]. Reagents
were added directed to a 96-well plate in the following order to
give a 100
peptide (200
ascorbate (10
m
L final solution: CuCl2 (400 nM), phosphate buffer, A
M), compounds (800 nM, 1% v/v DMSO), sodium
M). The reaction was allowed to incubate for 30 min
M of freshly pre-
b
Acknowledgments
m
m
This research work was financially supported by Innovative
Research Team in University (IRT1193), A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Edu-
cation Institutions (PAPD), Project of Graduate Education Innova-
tion of Jiangsu Province (No. CXZZ13_0320).
at room temperature. After this incubation, 50
m
pared working solution containing 100 nM Amplex Red (Sigma)
and 0.2 U/mL HRP (Sigma) in phosphate buffer was added to each
well, and the reaction was allowed to incubate for 30 min at room
temperature. Fluorescence was measured using a SpectraMax
Paradigm plate reader (lex
standard deviations for at least three measurements.
/
lem ¼ 530/590). Error bars represent
Appendix A. Supplementary data
Supplementary data related to this chapter can be found at
http://dx.doi.org/10.1016/j.ejmech.2014.10.004. These data include
MOL files and InChiKeys of the most important compounds
described in this article.
4.9. Cell culture and MTT assay for cell viability
The SH-SY5Y cells were cultured in Eagle's minimum essential
medium (EMEM)/ham's F-12 (1:1) medium supplemented with
10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL
streptomycin, at 37 ꢂC in a humidified atmosphere containing 5%
CO2. Cells were subcultured in 96-well plates at a seeding density of
1 ꢃ 104 cells/well and allowed to adhere and grow. When cells
reached the required confluence, they were placed into serum-free
medium and treated with compounds 2, 4, 11, 18, 22 and 23.
Twenty-four hours later the survival of cells was determined by
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