Synthesis of 2-pyridyl-benzimidazole iridium(III), ruthenium(II), and platinum(II) complexes. study of the activity as inhibitors of amyloid-β aggregation and neurotoxicity evaluation

Article abstract of DOI:10.1021/ic502119b

The design of small molecules that can target the aggregation of Aβ as potential therapeutic agents for Alzheimer's disease is an area of study that has attracted a lot of attention recently. The novel ligand methyl 1-butyl-2-pyridyl-benzimidazole carboxylate was prepared for the synthesis of a series of new iridium(III), ruthenium(II), and platinum(II) 2-pyridyl-benzimidazole complexes. The crystal structure of the half-sandwich iridium(III) complex was established by X-ray diffraction. An arrangement of two cationic complexes in the unit cell is observed, and it seems to be organized by weak ··· interactions that are taking place between two symmetry-related benzimidazole ring systems. All new compounds inhibited aggregation of Aβ1-42 in vitro as shown by both thioflavin T fluorescence assay and transmission electron microscopy. Among them the Ir compound rescued the toxicity of Aβ1-42 in primary cortical neurons effectively.

Full text of DOI:10.1021/ic502119b

Article
pubs.acs.org/IC
Synthesis of 2‑Pyridyl-benzimidazole Iridium(III), Ruthenium(II), and
Platinum(II) Complexes. Study of the Activity as Inhibitors of
Amyloid‑β Aggregation and Neurotoxicity Evaluation
†
†
†
‡
‡
,‡
Gorakh S. Yellol, Jyoti G. Yellol, Vijaya B. Kenche, Xiang Ming Liu, Kevin J. Barnham,*
§
,†
Antonio Donaire, Christoph Janiak, and Jose
́
Ruiz*
†
Departamento de Química Inorgan
́
ica and Regional Campus of International Excellence (Campus Mare Nostrum), Universidad de
Biosanitaria IMIB-Arrixaca, E-30071 Murcia, Spain
The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville 3016, Australia
Murcia and Instituto Murciano de Investigacion
́
‡
§
Institut fu
̈ ̈ ̈
̈
r Anorganische Chemie und Strukturchemie, Universitat Dusseldorf, D-40204 Dusseldorf, Germany
*
S Supporting Information
ABSTRACT: The design of small molecules that can target the aggregation of Aβ as
potential therapeutic agents for Alzheimer’s disease is an area of study that has
attracted a lot of attention recently. The novel ligand methyl 1-butyl-2-pyridyl-
benzimidazole carboxylate was prepared for the synthesis of a series of new
iridium(III), ruthenium(II), and platinum(II) 2-pyridyl-benzimidazole complexes.
The crystal structure of the half-sandwich iridium(III) complex was established by X-
ray diffraction. An arrangement of two cationic complexes in the unit cell is observed,
and it seems to be organized by weak π···π interactions that are taking place between
two symmetry-related benzimidazole ring systems. All new compounds inhibited
aggregation of Aβ1−42 in vitro as shown by both thioflavin T fluorescence assay and
transmission electron microscopy. Among them the Ir compound rescued the toxicity
of Aβ1−42 in primary cortical neurons effectively.
7
INTRODUCTION
oligomerization and attenuate its neurotoxicity in vitro.
■
Moreover, a few subsequent studies with other metal
compounds nicely corroborated the feasibility of such a
strategy. However, there are some critical factors that must
A century after the first report, the development of effective
treatment of Alzheimer disease (AD) remains challenging
4
(www.alz.org), and presently, there is no cure for this disease
be clarified for effective development of metal-based inhibitors
of β-amyloid aggregation. First of all, the evaluation of the
neurotoxicity of these compounds is strictly required with
respect to its ligand, metal, and their combination.
Subsequently, it would be extremely important to evaluate
the capability of metal compounds to cross the blood-brain
barrier.
that worsens progressively and eventually leads to death within
a few years. Also, for example, the health cost in the U.S. is
much higher for AD than it is for cardiovascular diseases and
cancer. Extracellular amyloid plaques with main constituent of
amyloid β-peptide (Aβ) and neurofibrillary tangles composed
of hyperphosphorylated tau protein are key pathological
1
indicators. Recent research proposes that the aggregation of
In this context, our study focused on evaluating the effect of
Aβ drives the disease process as various forms of aggregated Aβ
2
the type of metal on its ability to inhibit aggregation of Aβ and
have been revealed to be toxic. As a result, the development of
8
−11
its neurotoxicity.
Recently some platinum, ruthenium, and
a variety of therapeutic strategies targeting Aβ have been
stimulated. One approach is to design bifunctional molecules
that can chelate the metal out of Aβ and control or modulate its
iridium metal complexes have shown promising acivity as
4
,12−14
inhibitors of amyloid-β in their independent studies.
3
Therefore, it was determined to study the inhibition of
aggregation of Aβ1−42 in vitro of these precious metals
keeping the basic core ligand.
Here we disclose novel platinum, ruthenium, and iridium
benzimidazole complexes with activity as inhibitors of
aggregation of amyloid-β along with their toxicity profiles.
The designed concept of the present metal complexes has
aggregation. Another very different approach is to use metal
4
complexes that can modulate Aβ aggregation, Aβ containing a
metal-binding motif near the N terminus. Taking advantage of
this metal-binding ability, substantial efforts have been
dedicated to develop metal-based anti-Alzheimer compounds
5
,6
targeting Aβ. Metal−organic compounds could solve many of
the challenges in turning a structural lead into a drug candidate
with improved efficacy and tolerability as they are suitable for
rational drug design. In a pioneering study, Barnham et al.
proved that platinum complexes strongly inhibit amyloid
Received: August 31, 2014
©
XXXX American Chemical Society
A
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Inorganic Chemistry
Article
originated from our recent study of similar benzimidazole
anticancer compounds and the recognition of the biological
role of the benzimidazoles, which exhibit a wide range of
Scheme 1. Synthesis of Methyl 1-Butyl-2-pyridyl-
benzimidazole Carboxylate Pt(II)/Ru(II)/Ir(III) Complexes
15−22
pharmacological properties.
RESULTS AND DISCUSSION
Molecular Design. As shown in Figure 1, the typical active
N−N functionality embedded in pyridyl−benzimidazole will
■
Figure 1. Design of the 2-pyridyl-benzimidazole ligand for preparation
of metallodrugs.
serve as a basic core ligand with a readily available metalation
site. NH-functionality features an installation of different
moieties to modulate lipophilicity/hydrophilicity. The carboxyl
functionality is required to tune the design with the goal that it
serves as a handle for the intended functionalization of
metallodrugs with diverse moieties to explore their ability to
cross the blood-brain barrier.
including COSY, NOESY, and HSQC NMR techniques
(
Supporting Information, Figures S1−S3).
X-ray Crystal Structure of Complex 5·CH Cl . The
2
2
crystal structure of the iridium complex 5·CH Cl was
2
2
established by X-ray diffraction (Figure 2, Table 1, and
The designed molecule was synthesized using our developed
15
strategy reported earlier. Accordingly, key intermediate 1 was
synthesized from 4-fluoro-3-nitro-benzoic acid by acid catalyzed
methyl esterification, nucleophilic aromatic substitution of a
fluoro group by butyl amine, and subsequent reduction of a
nitro group using zinc and ammonium formate in methanol in
60% overall yield (Scheme 1). Aiming to tune the lipophilicity
of the final complex, a butyl group attached to the NH
functionality was the first choice. The core scaffold 2 was
synthesized by condensation of 1 and 2-pyridinecarboxaldehyde
in acidic ethanol at room temperature in good yield and
confirmed by spectroscopic methods (see Supporting Informa-
tion).
Synthesis of the New Metal Compounds. Compound 3
was prepared by treating pyridyl−benzimidazole 2 with an
equimolar mixture of Pt(DMSO) Cl (DMSO = dimethyl
2
2
sulfoxide) at room temperature for 24 h (Scheme 1).
Formation of complex 3 was confirmed by NMR spectroscopic
and electrospray ionization mass spectrometric (ESI-MS)
methods. Ligand 2 was also treated with para-cymene
ruthenium(II) [(p-cymene)RuCl2]2 in methanol at room
temperature to obtain, after addition of Bu NPF , the
Figure 2. X-ray crystallographic ORTEP diagram of the cation of
complex 5·CH Cl . Selected bond lengths (Å) and angles (deg): Ir−
2
2
Cl1 = 2.3929(4), Ir−N1 = 2.1208(15), Ir−N2 = 2.0809(14), Ir−
Cp(centroid) = 1.7903(8), Cl1−Ir−N2 = 85.32(4), Cl1−Ir−N1 =
85.93(4), N1−Ir−N2 = 75.08(6).
4
6
ruthenium cationic complex 4 in 72% yield. Formation of the
ruthenium complex was confirmed by spectroscopic methods.
In the H NMR spectrum of 4 introduction of four doublets at
Supporting Information, Tables S1−S4). Figure 2 depicts the
ORTEP diagram of the cation of complex 5, confirming its
“piano-stool” structure with the pentamethylcyclopentadienyl
1
6.5−6.7 ppm, a singlet at 2.2 ppm, and two doublets at 0.9 ppm
5
for six protons corresponding to p-cymene depicts the
formation of ruthenium complex 4. Similarly, the half-sandwich
iridium(III) complex 5 was prepared using a similar method
starting from the corresponding pentamethylcyclopentadienyl
chlorido iridium(III) dimer in good yield. Again formation of 5
was confirmed by spectroscopic and analytical methods,
group displaying the common π-bonded η coordination mode,
whereas the 1-butyl-2-pyridyl-benzimidazole carboxylate as-
2
sumes a bidentate-chelate coordination mode (κ -N,N), the two
rings of the benzimidazole and phenyl moieties being not
strictly coplanar. The Ir−N distance is shorter for the
benzimidazole heterocycle than it is for the pyridyl moiety.
B
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Inorganic Chemistry
Article
Table 1. Crystal Structure Determination Details of 5·
Biological Activity: Inhibition of Amyloid Aggrega-
tion Studies. All three compounds were tested for their ability
to inhibit amyloid aggregation of Aβ42 using a thioflavin T
fluorescence assay. The results indicated that 1 μM of all three
compounds was sufficient to inhibit Aβ amyloid aggregation
CH Cl2
2
5
·CH Cl
1
2
2
chemical formula
fw [g/mol]
cryst system
a [Å]
C H ClIrN O ·F P·CH Cl
28 34 3 2 6 2 2
902.13
(
Figure 4). Inhibition of amyloid formation was confirmed by
triclinic
transmission electron microscopy as shown in Figure 5.
8.3118(4)
11.8165(5)
16.9769(8)
91.208(1)
90.048(1)
100.290(1)
1640.21(13)
100(2)
b [Å]
c [Å]
α [deg]
β [deg]
γ [deg]
V [Å3]
temp (K)
space group
P1
̅
Z
2
μ [mm−1]
reflcns collcd
indpndnt reflcns
R(int)
4.43
65 066
10 041
0.039
0/430
1.059
restraints/params
GOF on F2
R1/wR2 [I > 2σ(I)]
R1/wR2 (all reflcns)
0.0184/0.0422
0.0203/0.0428
The complex 5 exhibits a CH Cl molecule (Supporting
2
2
Information, Figure S4) in the crystal lattice.
The unit cell of compound 5·CH Cl contains two molecular
2
2
units (Supporting Information, Figure S5). The overall crystal
packing is largely dominated by Coulomb interactions between
the (pentamethylcyclopentadienyl)-(methyl 1-butyl-2-pyridyl-
benzimidazole carboxylate)-chlorido-iridium(III) cation and
the PF anion. No C−H···π, and few significant C−H···Cl
6
and C−H···F interactions (Supporting Information, Table S5)
23
Figure 4. Inhibition of Aβ42 aggregation by (a) 3, (b) 4, and (c) 5.
Aβ42 (10 μM) was incubated at 37 °C in PBS buffer (pH 7.4) along
with varying concentrations of test compounds and ThT (20 μM).
Dose response curves representing Aβ42 alone (black), 20 μM (blue),
are indicated by PLATON. In view of the disorder in the PF6
anion (Supporting Information, Figure S6) we would, however,
not assign too much significance to the C−H···F interactions.
The arrangement of the two cations in the unit cell seems to be
organized by weak π···π interactions, which are depicted in
Figure 3 and Supporting Information, Figure S6 and are
detailed in Table S6 (Supporting Information). The π···π
interactions take place between two symmetry-related
1
0 μM (green), and 1 μM (red) were shown in the Tht assay (n = 2).
All the compounds showed inhibition of Aβ42 aggregation.
Rescue of Aβ Neurotoxicity. To ascertain whether the
inhibition of amyloid resulted in the rescue of Aβ neurotoxicity,
primary cortical neurons were treated with Aβ in the absence or
presence of the three diamagnetic compounds (Figure 6). At 10
μM Aβ on its own is toxic to the cells. Co-treatment with
compound 3 shows a marginal nonsignificant rescue of Aβ-
induced neurotoxicity; the labile compound 4 is itself toxic and
therefore unable to rescue Aβ toxicity. The more inert
compound 5 exhibited a robust rescuing of Aβ neurotoxicity.
A proof of the different kinetic behavior of the Ru and the Ir
compounds is that 4 (Ru) reacts readily with aqueous medium
2
4,25
benzimidazole ring systems.
1
as shown by H NMR (Supporting Information, Figure S7).
After the adequate functionalization to make complex 5 able to
cross the blood-brain barrier or to make complex 5 bioavailabe,
this iridium compound can be a good candidate for in vivo
studies.
Figure 3. π···π interactions with their centroid−centroid contacts
between two (pentamethylcyclopentadienyl)-(methyl 1-butyl-2-pyr-
idyl-benzimidazole carboxylate)-chlorido-iridium(III) cation in com-
pound 5·CH Cl .
CONCLUSIONS
We have successfully synthesized a novel methyl 1-butyl-2-
pyridyl-benzimidazole carboxylate ligand and a new series of
■
2
2
C
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Inorganic Chemistry
Article
Figure 5. Inhibition of Aβ42 aggregation: Electron micrographs of
Aβ42 (10 μM) incubated at 37 °C for 16 h, (a) Aβ42 alone, (b) with
complex 3 (10 μM), (c) with complex 4 (10 μM), (d) with complex 5
(
10 μM).
novel 2-pyridyl-benzimidazole precious metal complexes. The
crystal structure of the half-sandwich complex 5 shows that the
Ir−N distance is shorter for the benzimidazole heterocycle than
it is for the pyridyl moiety. Also weak π···π interactions are
observed that are taking place between two symmetry-related
benzimidazole ring systems. All new compounds inhibited
aggregation of Aβ1−42 in vitro as shown by both thioflavin T
fluorescence assay and transmission electron microscopy. Ir
complex 5 rescued the toxicity of Aβ1−42 in primary cortical
neurons effectively.
Figure 6. Inhibition of Aβ42 toxicity. Cell viability of primary cortical
neurons treated with Aβ42 for 4 d was measured by MTS absorbance
at 490 nm. (a) Aβ treatment reduces cell viability that is rescued by
coincubation of Aβ42 with complex 3. (b) Complex 4 itself is toxic to
the cells, and its coincubation with Aβ42 resulted in a negative effect.
(c) Complex 5 showed rescue of cell death from Aβ42 toxicity. “Media
only” is the DMSO negative control.
EXPERIMENTAL SECTION
Instrumental Measurements. The C, H, and N analyses were
■
1
performed with a Carlo Erba model EA 1108 microanalyzer. The H
and 13C NMR spectra were recorded on a Bruker AC 300E or a Bruker
1
AV 400 spectrometer. Chemical shifts are cited relative to SiMe ( H
4
and 13C, external). ESI mass (positive mode) analyses were performed
on a HPLC/MS TOF 6220. UV−vis spectroscopy was carried out on a
PerkinElmer Lambda 750 S spectrometer with operating software.
Fluorescence measurements were carried out with a PerkinElmer LS
(butylamino)-3-nitrobenzoate in 79% yield. Subsequently, methyl 4-
(butylamino)-3-nitrobenzoate (1 mmol) was dissolved in methanol
(10 mL) in a round-bottom flask equipped with a stirrer and nitrogen
atmosphere. Zinc (3 mmol) was added at room temperature with
constant stirring followed by addition of ammonium format (2 mmol)
in two batches. The reaction mixture was stirred at room temperature
for 5 h, and the progress of reaction was monitored by TLC. After
complete conversion, reaction was filtered to remove zinc and
unreacted ammonium formate. Filtrate was concentrated and then
dissolved in dichloromethane and stirred for 30 min. The undissolved
material was removed by filtration, and dichloromethane was
concentrated under reduced pressure. The crude product was purified
by column chromatography using ethyl acetate−hexane (1:2) as eluent
to get methyl 3-amino-4-(butylamino)benzoate (1) in 68% yield.
Preparation and Characterization of Compound 2. Methyl 3-
amino-4-(butylamino)benzoate 1 (1.0 mmol) was dissolved in ethanol
(10 mL) in round-bottom flask equipped with stirrer and nitrogen
atmosphere. 2-Pyridinecarboxaldehyde (1.0 mmol) was added at room
temperature with constant stirring followed by addition of trifluoro-
acetic acid (0.1 mmol) and magnesium sulfate (5 mmol). The reaction
5
5 50 Hz Fluorescence Spectrometer.
Materials. Solvents were dried by the usual methods. K PtCl ,
2
4
6
5
[
(η -cymene)RuCl ] , [(η -C Me )IrCl ] , were obtained from Sigma-
2 2 5 5 2 2
Aldrich (Madrid, Spain); Pt(DMSO) Cl was prepared using reported
2
2
26
procedure.
Preparation of Compound 1. Methyl 4-fluoro-3-nitrobenzoate
1 mmol) was dissolved in dichloromethane (10 mL) in round-bottom
(
flask equipped with stirrer and nitrogen atmosphere. Butyl amine (2
mmol) was added to it at room temperature with constant stirring
followed by addition of triethylamine (2 mmol). The reaction mixture
was stirred at room temperature for 12 h, and the progress of reaction
was monitored by thin-layer chromatography (TLC). After complete
conversion, reaction was quenched by water (10 mL), and product was
extracted by dichloromethane (2 × 10 mL). The combined
dichloromethane layer was washed with water (10 mL) and brine
(
10 mL), dried on sodium sulfate, and concentrated under reduced
pressure. The crude product was purified by column chromatography
using ethyl acetate−hexane (1:5) as eluent to get methyl 4-
D
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Inorganic Chemistry
Article
mixture was stirred at room temperature for 24 h, and the progress of
reaction was monitored by TLC. After complete conversion, reaction
was filtered to remove magnesium sulfate. Filtrate was concentrated
and then dissolved in dichloromethane. Dichloromethane was washed
with water (2 × 10 mL) and brine (10 mL), dried on sodium sulfate,
and concentrated under reduced pressure. The crude product was
purified by column chromatography using ethyl acetate−hexane (1:3)
as eluent to obtain methyl 1-butyl-2-(pyridin-2-yl)-1H-benzo[d]-
imidazole-5-carboxylate (2) in 57% yield. Mr (C H N O ) =
temperature for the next 15 h. The yellow solution was concentrated
to 15 mL, and Bu NPF (1.0 mmol) was added to it. The reaction
4
6
mixture was stirred at room temperature for the next 30 min to furnish
yellow precipitate, which was filtered and washed with methanol and
diethyl ether. The yellow crystalline iridium complex 5 was obtained in
good yield (87%). M (C H ClF N O PRu) = 817,1611 g/mol. Anal.
r
28 33
6
3
2
Calcd for C H ClF IrN O P: C, 41.15; H, 4.19; N, 5.14; Found: C,
2
8
34
6
3
2
1
41.34; H, 4.07; N, 5.20%. H NMR (400.13 MHz, CDCl ) δ 8.98 (d, J
3
18
19
3
2
= 5.0 Hz, 1H), 8.55 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.21−8.25 (m,
3
09.1477 g/mol. Anal. Calcd for 2 C H N O : C, 69.88; H, 6.19;
1H), 7.73 (t, J = 6.4 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 4.94−5.02 (m,
18 19 3 2
1
N, 13.58. Found: C, 70.12; H, 6.56; N, 13.27%. H NMR (400.13
1
H), 4.63−4.71 (m, 1H), 4.02 (s, 3H), 1.94−1.97 (m, 2H), 1.77 (s,
13
MHz, CDCl ) δ 0.94 (t, 3H, J = 7.3 Hz), 1.32−1.42 (m, 2H), 1.82−
3
15H), 1.53−1.58 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H); C NMR (100.60
MHz, CDCl ) δ 166.0, 152.6 (2C), 145.8, 139.2, 137.4 (2C), 128.4,
1
1
.92 (m, 3H), 3.96 (s, 3H), 4.07 (t, 2H, J = 7.2 Hz), 7.38−7.42 (m,
H), 7.50 (d, 1H, J = 8.5 Hz), 7.90 (m, 1H), 8.08 (dd, 1H, J = 1.5 Hz,
3
1
1
27.4 (2C), 127.2, 125.1, 119.7, 112.2, 89.2 (5C), 52.6, 31.5, 20.0,
J = 7.0 Hz), 8.52 (d, 1H, J = 7.8 Hz), 8.60 (s, 1H), 8.72 (d, 1H, J = 4.4
3.5, 9.2 (5C); Positive-ion ESI mass spectra ion cluster (DMSO) at
Hz); 13C NMR (100.60 MHz, CDCl ) δ 167.5, 151.5, 150.2, 148.6,
+
3
m/z = 672.1938 [M−PF ] .
6
1
3
3
42.1, 139.8, 136.8, 124.8, 124.5 (2 C), 124.0, 122.4, 109.8, 52.0, 45.5,
X-ray Crystal Structure Analysis. Suitable crystals of 5 were
grown from dichloromethane/hexane. Crystal data, data collection,
and structure refinement details are summarized in Supporting
Information, Table S1. Hydrogen atoms for aromatic CH, aliphatic
2.1, 19.9, 213.6; Positive-ion ESI mass spectra (DMSO) at m/z =
+
10.1552 [M + H] .
Preparation and Characterization of Compound 3. Methyl 1-
butyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxylate 2 (1
mmol) was dissolved in freshly distilled dichloromethane in a dry
round-bottom flask equipped with stirrer and nitrogen atmosphere.
Sodium acetate (1.2 mmol) was added to this at room temperature
with constant stirring followed by addition of Pt(DMSO) Cl (0.5
CH, CH , and methyl groups were positioned geometrically (C−H =
2
0
0
.95 Å for aromatic CH, C−H = 1−00 Å for aliphatic CH, C−H =
.99 Å for CH , C−H = 0.98 Å for CH ) and refined using a riding
2
3
model (AFIX 43 for aromatic CH, AFIX 23 for CH , AFIX 137 for
2
2
2
rotating group for CH ), with U (H) = 1.2U (CH) and U (H) =
3
iso
eq
iso
mmol). The reaction mixture was stirred at room temperature for 24
h. The precipitate formed was filtered and washed with dichloro-
methane and diethyl ether, respectively. The yellowish color platinum
complex 3 was obtained in good yield (77%). M (C H Cl N O Pt)
1
.5U (CH ).
eq 3
The PF anion is rotationally disordered around the F5−P−F6 axis
6
(
see Supporting Information, Figure S4). Two sets of F1−F4 positions
r
18 19
2
3
2
in the square plane could be refined with PART commands. The major
component F1−F4 has an ∼80% occupation, while F1b−F4b are
occupied to 20%. The minor F1b−F4b were refined isotropically.
In Situ ThT Fluorescence Assay for Aβ Aggregation.
Compounds to be tested were dissolved at 400, 200, and 20 μM in
=
574.0502 g/mol. Anal. Calcd for C H Cl N O Pt: C, 37.58; H,
18 19 2 3 2
1
3
.33; N, 7.30; Found: C, 37.88; H, 3.50; N, 7.41%. H NMR (400.13
MHz, CDCl ) δ 9.64−9.66 (m, 2H), 8.45 (dt, 1H, J = 8.0, 1.4 Hz),
3
8
4
1
.35 (d, 1H, J = 8.0 Hz), 8.10−8.11 (m, 2H), 7.85 (t, 1H, J = 7.2 Hz),
.83 (t, 1H, J = 7.4 Hz), 3.90 (s, 3H), 1.85 (quint, 2H, J = 7.4 Hz),
1
00% DMSO stock solution. The final DMSO concentration was 5%.
13
.44−1.39 (m, 2H), 0.89 (t, 3H, J = 7.4 Hz); C NMR (100.60 MHz,
Each sample (500 μL) was prepared using the appropriate
concentration of compound (25 μL), Aβ42 (100 μM of 50 μL), and
ThT (20 μM of 425 μL) and pipetted out in 150 μL aliquots into
three wells of black bottom clear 96-microwell plate. The plate was
sealed to prevent evaporation and was put into the plate reader (BMG
Labtechnologies), which was then set to incubate at 37 °C for 16 h.
The ThT fluorescence was recorded at every 15 min (with 10 s of
orbital shaking) interval via bottom reading with excitation at 440 and
emission at 490 nm.
CDCl ) δ 199.90, 157.41, 150.02, 141.53, 138.54, 137.85, 137.26,
3
1
2
25.27, 124.98, 124.29, 120.90, 118.61, 115.12, 51.53, 49.44, 33.15,
0.16, 13.87; Positive-ion ESI mass spectra ion cluster (DMSO) at m/
+
z = 568.0857 [M + H O] .
2
Preparation and Characterization of Compound 4. Methyl 1-
butyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxylate 2 (1.0
mmol) was dissolved in freshly distilled methanol (30 mL) in a dry
round-bottom flask equipped with stirrer and nitrogen atmosphere.
6
[
(η -cymene)RuCl ] (0.5 mmol) was added at room temperature
2
2
Electron Microscopy. Electron microscopy was performed to
confirm the effect of test compounds on formation of any aggregates/
fibrils and to observe any possible morphological changes. Aliquots
were collected from the assay samples at the end of incubation for
imaging. EM carbon-coated gold grids (ProSciTech, Kirwan, Australia)
were glow-discharged (Brodsky et al., 1977) and coated (carbon-
coated side up) with 20 μM of assay samples. The grids were then
blotted with filter paper to remove excess solvent, washed quickly with
with constant stirring. The reaction mixture was stirred at room
temperature for the next 15 h. The yellow solution was concentrated
to 15 mL, and Bu NPF (1.0 mmol) was added in it. The reaction
4
6
mixture was stirred at room temperature for the next 30 min to furnish
yellow precipitate, which was filtered and washed with methanol and
diethyl ether. The yellow crystalline ruthenium complex 3 was
obtained in good yield (85%). M (C H ClF N O PRu) = 725.0947
r
28 33
6
3
2
g/mol. Anal. Calcd for C H ClF N O PRu: C, 46.38; H, 4.59; N,
2
8
33
6
3
2
1
10 μL of mH O, blotted, and negatively stained with 10 μL of 1.5%
5
.80; Found: C, 46.71; H, 4.33; N, 6.03%. H NMR (400.13 MHz,
2
(w/v) uranyl acetate (UA) for 20 s. Grids were then blotted again and
CDCl ) δ 0.96 (t, 3H, J = 7.3 Hz), 1.05−1.08 (m, 6H), 1.42−1.49(m,
3
air-dried before analysis. Images were taken using a Tecnai G2
transmission electron microscope (TEM) (FEI, Hillsboro, OR, U.S.)
operating at 200 kV.
Aβ42 Neurotoxicity Rescue. Aβ42 Preparation. Aβ42 (pur-
chased from Keck lab) treated with HFIP (hexafluoroisopropenol,
Sigma 105228) followed by air-dry with speed Vac and storage at −20
°C. Dissolved HFIP treated Aβ42 with 10% of 20 mM of NaOH in
phosphate buffered saline (PBS) and determined Aβ42 concentration
by measuring the absorbance at λ 214 nm.
Primary Cortical Neuron Preparation. Removed fetuses from
pregnant C57BL/6 pregnant mouse at gestation (14 d) and isolated
primary cortical neurons. Place 150 000 neuronal cells to each well in a
48-well plate (pretreated with Poly-D-Lysine) in grow media of
Neurobasal media supplement with B27 supplement, Gentamycin, and
glutamax (all from Life Technologies) and incubated the cells at 37 °C
2
3
H), 1.86−1.96 (m, 2H), 2.22 (s, 3H), 2.62−2.71 (m, 1H), 4.05 (s,
H), 4.59−4.83 (m, 2H), 5.82 (d, 1H, J = 6.5 Hz), 5.90 (d, 1H, J = 6.5
Hz), 6.03 (d, 1H, J = 5.5 Hz), 6.10 (d, 1H, J = 5.9 Hz), 7.67−7.70 (m,
2
8
H), 8.14−8.23 (m, 2H), 8.26−8.29 (dd, 1H, J = 1.5 Hz, J = 7.3 Hz),
.54 (s, 1H), 9.54 (d, 1H, J = 5.5 Hz); 13C NMR (100.60 MHz,
CDCl ) δ 166.02, 157.47, 150.11, 145.11, 140.17, 139.86, 139.02,
3
1
28.08, 127.82, 127.49, 124.62, 119.90 (4C), 112.02, 85.94, 84.20,
8
3.18, 81.29, 52.72, 46.23, 31.51, 31.20, 21.99, 19.90, 18.75, 13.52;
Positive-ion ESI mass spectra ion cluster (DMSO) at m/z = 580.1308
+
[
M−PF ] .
6
Preparation and Characterization of Compound 5. Methyl 1-
butyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxylate 2 (1
mmol) was dissolved in freshly distilled methanol (30 mL) in a dry
round-bottom flask equipped with stirrer and nitrogen atmosphere.
5
[
(η -C Me )IrCl ] (0.5 mmol) was added at room temperature with
5
5
2 2
constant stirring. The reaction mixture was stirred at room
in the chamber (5% CO ) for 6 d.
2
E
dx.doi.org/10.1021/ic502119b | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Assay. Primary cortical neurons were treated with Aβ42 10 μM
final concentration) and test compounds at 10, 5, 2.5, and 1.25 μM
final concentration) in growth media of Neurobasal media supple-
(13) He, L.; Wang, X.; Zhao, C.; Wang, H.; Du, W. Metallomics 2013,
5, 1599.
(14) Collin, F.; Sasaki, I.; Eury, H.; Faller, P.; Hureau, C. Chem.
(
(
ment with B27 AO supplement. The cells treated with Aβ42 were
taken as positive control, the cells treated with DMSO were taken as
negative control, and the cells treated with compounds only were
taken as negative control. Cells were incubated at 37 °C in 5% CO2
chamber for 4 d. Cell viability was determined using cell counting kit
Commun. 2013, 49, 2130.
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2014, 57, 2498.
(
CCK-8) assay after 4 d of treatment. The data were normalized and
calculated as a percentage of untreated control values. Each treatment
group was done in triplicate.
(17) Schiffmann, R.; Neugebauer, A.; Klein, C. D. J. Med. Chem.
2006, 49, 511.
(
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I.; Waldmann, H. Nature 2013, 497, 638.
ASSOCIATED CONTENT
■
(
*
S
Supporting Information
Structures, energies and Cartesian coordinates for all computed
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org. CCDC deposition number
(
20) Ong, J. X.; Yap, C. W.; Ang, W. H. Inorg. Chem. 2012, 51,
12483.
(21) Ginzinger, W.; Mu
1
012641.
̈
hlgassner, G.; Arion, V. B.; Jakupec, M. A.;
Roller, A.; Galanski, M.; Reithofer, M.; Berger, W.; Keppler, B. K. J.
Med. Chem. 2012, 55, 3398.
AUTHOR INFORMATION
■
*
(
22) Noel
̈
, S.; Cadet, S.; Gras, E.; Hureau, C. Chem. Soc. Rev. 2013,
Corresponding Authors
Fax: +61393476750. Phone:+61383442555. E-mail:
kbarnham@unimelb.edu.au.
Fax: +34868884148. Phone: +34868887455. E-mail: jruiz@
4
2, 7747.
(23) (a) Spek, A. Acta Crystallographica Section D 2009, 65, 148−155.
(b) Spek, A. L. PLATON − A multipurpose crystallographic tool;
*
Utrecht University: Utrecht, The Netherlands, 2005.
(24) Janiak, C. J. Chem. Soc., Dalton Trans. 2000, 3885.
um.es.
(25) Yang, X. J.; Drepper, F.; Wu, B.; Sun, W. H.; Haehnel, W.;
Notes
Janiak, C. Dalton Trans. 2005, 256. and supplementary material
therein..
The authors declare no competing financial interest.
(
26) Price, J. H.; Williamson, A. N.; Schramm, R. F.; Wayland, B. B.
ACKNOWLEDGMENTS
Inorg. Chem. 1972, 11, 1280.
■
This work was financially supported by the European Union
Seventh Framework Programme−Marie Curie Cofund (FP7/
2
007−2013) under U-IMPACT Grant No. 267143 and the
́
Spanish Ministerio de Economia y Competitividad and FEDER
(
Project No. SAF2011-26611), the Fundacion
́ ́
Seneca-CARM
(Project 15354/PI/10) and COST CM1105 for providing
opportunities of discussion.
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dx.doi.org/10.1021/ic502119b | Inorg. Chem. XXXX, XXX, XXX−XXX