Importance of the Dimethylamino Functionality on a Multifunctional Framework for Regulating Metals, Amyloid-β, and Oxidative Stress in Alzheimers Disease

Article abstract of DOI:10.1021/acs.inorgchem.6b00525

The complex and multifaceted pathology of Alzheimers disease (AD) continues to present a formidable challenge to the establishment of long-term treatment strategies. Multifunctional compounds able to modulate the reactivities of various pathological featu

Full text of DOI:10.1021/acs.inorgchem.6b00525

Article
pubs.acs.org/IC
Importance of the Dimethylamino Functionality on a Multifunctional
Framework for Regulating Metals, Amyloid-β, and Oxidative Stress in
Alzheimer’s Disease
†
‡
‡
‡
†
†
Jeffrey S. Derrick, Richard A. Kerr, Kyle J. Korshavn, Michael J. McLane, Juhye Kang, Eunju Nam,
Ayyalusamy Ramamoorthy,‡,§ Brandon T. Ruotolo, and Mi Hee Lim*
‡
,†
†
Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
‡
§
Departments of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States
*
S Supporting Information
ABSTRACT: The complex and multifaceted pathology of
Alzheimer’s disease (AD) continues to present a formidable
challenge to the establishment of long-term treatment strategies.
Multifunctional compounds able to modulate the reactivities of
various pathological features, such as amyloid-β (Aβ) aggregation,
metal ion dyshomeostasis, and oxidative stress, have emerged as a
useful tactic. Recently, an incorporation approach to the rational
design of multipurpose small molecules has been validated
through the production of a multifunctional ligand (ML) as a
potential chemical tool for AD. In order to further the
development of more diverse and improved multifunctional
reagents, essential pharmacophores must be identified. Herein,
we report a series of aminoquinoline derivatives (AQ1−4, AQP1−4, and AQDA1−3) based on ML’s framework, prepared to
gain a structure−reactivity understanding of ML’s multifunctionality in addition to tuning its metal binding affinity. Our
structure−reactivity investigations have implicated the dimethylamino group as a key component for supplying the
antiamyloidogenic characteristics of ML in both the absence and presence of metal ions. Two-dimensional NMR studies indicate
that structural variations of ML could tune its interaction sites along the Aβ sequence. In addition, mass spectrometric analyses
suggest that the ability of our aminoquinoline derivatives to regulate metal-induced Aβ aggregation may be influenced by their
metal binding properties. Moreover, structural modifications to ML were also observed to noticeably change its metal binding
affinities and metal-to-ligand stoichiometries that were shown to be linked to their antiamyloidogenic and antioxidant activities.
Overall, our studies provide new insights into rational design strategies for multifunctional ligands directed at regulating metal
ions, Aβ, and oxidative stress in AD and could advance the development of improved next-generation multifunctional reagents.
INTRODUCTION
(e.g., cholinergic, glutamatergic, and dopaminergic receptors),
and enzymes (e.g., acetylcholinesterase, monoamine oxi-
■
(
Effective therapeutic strategies to combat Alzheimer’s disease
AD), the most common form of dementia, have yet to be
identified, which is most likely a result of the disease’s complex
and multifaceted pathology.
1
,15−22
dase).
Unfortunately, a majority of the current multi-
functional molecules are developed by slightly modifying
1
−10
existing drugs or known molecular scaffolds (e.g., tacrine,
For example, some patho-
1
,15−22
coumarin, curcumin).
Such a tactic is often costly and
logical features being actively investigated include: misfolded
and aggregated proteins (i.e., amyloid-β (Aβ) and tau), metal
ion dyshomeostasis and miscompartmentalization, oxidative
stress, excitotoxicity, neuroinflammation, and mitochondrial
time-consuming since it frequently involves the high-
throughput screening of many structural derivatives, most of
which fail to be selected for further analysis.
1
−6,11,12
Rather than modifying familiar frameworks, we have recently
reported that novel multifunctional AD agents can be generated
through the use of an incorporation approach to rational ligand
damage.
Furthermore, the interconnections between
many of these pathological factors, such as Aβ, metal ions, and
reactive oxygen species (ROS), make the elucidation of a
comprehensive molecular-level understanding of AD etiology
23,24
design.
Initial studies with a multifunctional ligand (ML)
1
−4,6−9,13,14
identified the feasibility of designing a single molecular entity
that can control metal-free and metal-induced Aβ aggregation,
toxicity engendered by metal-free Aβ and metal−Aβ, and
extremely challenging.
One approach to address the
inherently complex multifaceted nature of AD is to utilize
multifunctional compounds able to preferentially modulate the
activities of multiple targets simultaneously. This strategy has
been increasing in prevalence within the literature with
common targets, including Aβ, tau, various neuroreceptors
Received: March 2, 2016
©
XXXX American Chemical Society
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Figure 1. Structural variations on a multifunctional ligand (ML) framework. Modifications were performed on the multimodal scaffold to identify a
structure−reactivity understanding of ML’s multifunctionality as well as to tune its metal binding characteristics. The quinoline portion of the
structure was examined by cleavage of the HN−CH bond (site 1, purple) and modulation of the functionalities at the R position (site 3, blue).
2
2
Furthermore, simultaneous structural alterations at the R and R positions (sites 2 and 3, green and blue, respectively) allowed the role of the
1
2
dimethylamino group and the 4-(dimethylamino)phenol in ML’s activities to be illuminated.
a
Table 1. Values (MW, clogP, HBA, HBD, PSA, logBB, and −logP ) of AQDA1−3 and ML
e
b
Calculation
MW
AQDA1
AQDA2
AQDA3
ML
Lipinski’s rules and others
293
3.15
307
3.65
351
2.91
396
2.57
≤ 450
≤ 5.0
≤ 10
≤ 5
clogP
HBA
HBD
PSA
4
4
6
5
2
2
2
3
2
48.4
48.4
74.7
68.6
≤ 90 Å
logBB
−0.107
4.30 ± 0.01
CNS+
−0.323
4.30 ± 0.01
CNS+
−0.533
4.30 ± 0.01
CNS+
−0.496
4.50 ± 0.01
CNS+
< −1.0 (poorly distributed in the brain)
−
logPe
−logP < 5.4 (CNS+); − logP > 5.7 (CNS−)
e
e
CNS±prediction
CNS+; CNS−
a
MW, molecular weight; clogP, calculated logarithm of the octanol-water partition coefficient; HBA, hydrogen bond acceptor atoms; HBD, hydrogen
bond donor atoms; PSA, polar surface area; logBB = −0.0148 × PSA + 0.152 × clogP + 0.139 (logBB < −1.0 poorly distributed in the brain); −logPe
values, determined using the PAMPA-BBB assay, were calculated by the PAMPA 9 explorer software V. 3.5. Prediction of compound’s ability to
penetrate the central nervous system (CNS) on the basis of literature values. Compounds categorized as CNS+ possess the potential ability to
b
penetrate the BBB while those categorized as CNS− are expected to have poor BBB permeability. Reference 23.
metal-mediated ROS generation, as well as scavenge free
binding site are also indicated to direct the derivatives’ ability to
bind Cu(II) and Zn(II), control reactive oxygen species (ROS)
formation, and alter Cu(II)−/Zn(II)−Aβ aggregation. The
overall structural variation of ML also tuned its capability to
scavenge free radicals. Mass spectrometric studies further
illustrate the importance of the metal binding affinity of this
series of small molecules in regulating metal−Aβ aggregation
and potentially suggest larger, higher-order oligomers as the
interacting species. Similar to ML, our structural derivatives are
also observed to be potentially suitable in biological systems
since they are moderately water soluble and potentially possess
the ability to penetrate the blood-brain barrier (BBB). Overall,
our studies highlight the importance of the dimethylamino
moiety for imparting reactivity toward AD-relevant targets (e.g.,
metals, Aβ, metal−Aβ), yet further studies are still warranted to
assess its transferability to other molecular scaffolds.
radicals, overall validating ML’s structure-based design
1
2,23,24
strategy.
Further progress toward the production of
more diverse and improved multifunctional reagents is
dependent on the recognition of critical pharmacophores that
can be employed as figurative “building blocks” to engineer
next-generation ligands through such a rational design
approach. Structure−reactivity studies, using individual compo-
nents within a complex molecule and structurally modified
molecules, can provide the information to determine the
chemical functionalities that may impart the desired reactivities.
Toward this goal, we have prepared a series of aminoquino-
line (AQ) derivatives (i.e., AQ1−4, AQP1−4, AQDA1−3;
Figure 1) based on the framework of ML in order to discern a
structure−reactivity understanding of ML’s multifunctionality.
Our in vitro investigations have proposed the dimethylamino
functionality of ML to be critical toward its ability to modulate
the aggregation pathways of metal-free Aβ and metal−Aβ.
Two-dimensional (2D) NMR studies have shown that
functionalization of the aminoquinoline moiety is capable of
shifting the preferred region of interaction along the sequence
of Aβ. In addition, the slight modifications to ML’s metal
RESULTS AND DISCUSSION
■
Design Rationale and Preparation for Structural
Modifications to a Multifunctional Framework. A series
of aminoquinoline (AQ) derivatives based on our previously
reported multifunctional ligand (ML) were generated to (i)
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develop a structure−reactivity understanding of ML’s multi-
faceted reactivity toward metal-free Aβ, metal−Aβ, ROS, and
free radicals, and (ii) reduce its relatively high binding affinity
for Cu(II) and Zn(II) (i.e., dissociation constants for Cu(II)
and Zn(II) in the picomolar and nanomolar range, respectively)
that may potentially interfere with biologically essential
metalloproteins. The AQ derivatives were obtained by
subjecting ML to modifications at three sites (Figure 1).
First, in order to determine the significance of the phenol or 4-
Scheme 1. Synthetic Routes to AQ, AQP, and AQDA
Derivatives
2
3
(
dimethylamino)phenol moieties in ML’s activities, AQ1−4
were prepared by excision of the HN−(CH ) bond (site 1,
3
2
Figure 1). The role of the dimethylamino moiety in directing
ML’s multifunctionality was also evaluated by modifying site 2
through installation of a hydrogen atom (AQP1−4) or a
dimethylamino group (AQDA1−4) (Figure 1). Further
modifications to the metal binding site (site 3) from a
hydrogen atom (in AQ1, AQP1, and AQDA1) or a methyl
group (in AQ2, AQP2, and AQDA2) to an ester (in AQ3,
AQP3, and AQDA3) or an alcohol moiety (in AQ4, AQP4,
and ML) allowed us to examine the difference in denticity and
electronics of the ligands (Figure 1). Similar to ML, all AQ
derivatives were also designed to adhere to Lipinski’s rules and
calculated logBB values for potential drug-likeness and BBB
assemble into a distribution of large aggregates that are too big
to penetrate into the gel matrix, which yields very little
smearing in the gel/Western blots, but can be visualized via
23,25,26
23,31,32
permeability (Table 1 and Table S1).
TEM.
The administration of compounds, capable of
Synthetically, the structural derivatives were obtained via
synthetic routes analogous to the one previously reported for
interacting with Aβ, inhibiting the formation of high MW
aggregates, and/or disassembling preformed aggregates,
typically generates a distribution of smaller-sized Aβ species
that can enter into the gel and produce a substantial amount of
2
3,27−30
ML.
Starting from 2-methyl-8-nitroquinoline, the AQ
derivatives, AQ3 and AQ4, were produced through a multistep
31
23,31,32
reaction (AQ1 and AQ2 were commercially available). First,
the nitro precursor compound, methyl 8-nitroquinoline-2-
carboxylate, was prepared by bromination of 2-methyl-8-
nitroquinoline followed by hydrolysis in 20% sulfuric acid to
afford 8-nitroquinoline-2-carboxylic acid. 8-Nitroquinoline-2-
carboxylic acid was then methylated with trimethylsilyldiazo-
methane (Me SiCHN ), a safer alternative to diazomethane, to
streaking compared to the samples containing only Aβ.
In the inhibition experiments, only the derivatives containing
the 4-(dimethylamino)phenol moiety (i.e., AQDA1−3) were
able to modulate the MW distribution of metal-free Aβ40 and
Zn(II)−Aβ40 (Figure 2b, third column, lanes 1−3). In the case
of Cu(II)−Aβ40, only one compound without the 4-
(dimethylamino)phenol, AQP1, in addition to AQDA1−3,
produced detectable smearing in the high MW region of the
gel/Western blot (>100 kDa; Figure 2b, second column, lane
1). The reactivity of AQP1 for Cu(II)−Aβ40 may be a result of
its relatively high binding affinity for Cu(II) compared to that
of the other multifunctional derivatives (vide infra). The
inhibitory reactivity of AQDA1−3 toward metal-free and metal-
treated Aβ40 also appeared to be time dependent. Longer,
darker bands (ca. 4−260 kDa) were detected on the gel/
Western blot following later incubation periods (i.e., 24 h) of
metal-free Aβ40 and metal−Aβ40 with AQP1 or AQDA1−3
(Figure 2b).
3
2
produce the precursor (i.e., methyl 8-nitroquinoline-2-carbox-
ylate) to AQ3 and AQ4. Hydrogenation of methyl 8-
nitroquinoline-2-carboxylate in the presence of 10% palladium
on carbon provided AQ3 at a modest yield (ca. 50%). Further
reduction of AQ3 with sodium borohydride (NaBH4)
generated AQ4. The derivatives containing an aminoquinoline
and phenol (i.e., AQP1−4) or a 4-(dimethylamino)phenol (i.e.,
AQDA1−3) were constructed by a Schiff base condensation
reaction of AQ1−4 with either salicylaldehyde (for AQP1−3)
or 4-(dimethylamino)-2-hydroxybenzaldehyde (for AQDA1−
3) followed by the reduction of the resultant imine with sodium
triacetoxyborohydride as shown in Scheme 1.
AQ derivatives also had a similar aptitude for inhibiting the
self-assembly of the more aggregation-prone isoform,
Effects of AQ Derivatives on Metal-Free and Metal-
Induced Aβ Aggregation. In order to determine the effect of
structural modifications on the ability of AQ derivatives to
modulate Aβ aggregation in both the absence and presence of
metal ions [Cu(II) and Zn(II)], gel electrophoresis with
Western blotting (gel/Western blot) utilizing an anti-Aβ
antibody (6E10) and transmission electron microscopy
6
,11,12
Aβ42.
Only AQDA1−3 with the 4-(dimethylamino)-
phenol functionality perturbed the MW distribution of the
resultant metal-free and metal-induced Aβ42 aggregates
different from that of the control samples (Figure 3b, third
column, lanes 1−3). Unlike in the Aβ40 conditions, AQP1 was
not indicated to significantly ameliorate the aggregation of
Cu(II)−Aβ42, which might be a result of the faster aggregation
thus limiting the interaction with Cu(II) surrounded by Aβ42
(Figure 3b, second column, lane 1). TEM images of metal-free
and metal-bound Aβ40 and Aβ42 samples treated with
AQDA1−3 revealed a shift from the large Aβ aggregates and
fibrils found in the compound-untreated samples of metal-free
and metal-bound Aβ toward morphologies that are much
smaller and more amorphous (Figure 4 for Aβ42; Figure S3 for
Aβ40). Consistent with the gel/Western blot findings, the
(
(
TEM) were performed to analyze the molecular weight
MW) distribution and morphological change of the resultant
Aβ species, respectively. Two experiments were conducted to
determine (i) the ability of the derivatives to prevent the
formation of fibrillar aggregates (inhibition experiment, Figures
2a and 3a) and (ii) to dismantle preformed Aβ aggregates into
smaller species (disaggregation experiment, Figures S1a and
S2a). Generally, under the experimental conditions employed
herein, compound-free Aβ samples with and without metal ions
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Figure 2. Ability of compounds (AQ1−3, AQP1−4, and AQDA1−3) to control the formation of Aβ40 aggregates in the absence and presence of
metal ions [Cu(II) and Zn(II)]. (a) Scheme of the inhibition experiments. (b) Visualization of the resultant Aβ species from the inhibition
experiments by gel/Western blot utilizing an anti-Aβ antibody (6E10). The control lane (without compound treatment) is identified by the letter C,
and the lane number refers to the specific compound within each small molecule group (i.e., AQ, AQP, AQDA).
Figure 3. Capability of compounds (AQ1−3, AQP1−4, and AQDA1−3) to inhibit the formation of Aβ42 aggregates in the absence and presence of
metal ions [Cu(II) and Zn(II)]. (a) Scheme of the inhibition experiments. (b) Visualization of the resultant Aβ species from the inhibition
experiments by gel/Western blot utilizing an anti-Aβ antibody (6E10). The control lane (without compound treatment) is identified by the letter C,
and the lane number refers to the specific compound within each small molecule group (i.e., AQ, AQP, AQDA).
aminoquinoline derivative, AQ1, and aminoquinolinephenol
derivative, AQP1, were not observed to significantly alter the
size or morphology of metal-free Aβ or Zn(II)−Aβ (Figure 4
and Figure S3). Some smaller and more unstructured
aggregates, however, were identified in the Cu(II)−Aβ40
inhibition samples incubated with AQP1 (Figure S3, middle
column, inset).
We also evaluated the capacity of the multifunctional
derivatives to interact with and degrade preformed Aβ40 and
Aβ42 aggregates (i.e., disaggregation experiments; Figures S1a
and S2a). The disaggregation experiments showed trends
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Figure 4. Morphologies of the resultant metal-free Aβ42 and metal−Aβ42 aggregates upon treatment with AQ1, AQP1, and AQDA1−3. (a)
Scheme of the inhibition experiments. (b) TEM images for the Aβ42 samples (24 h incubation). Insets represent the minor species.
similar to those observed in the inhibition studies. Only
compounds with the 4-(dimethylamino)phenol moiety
the N-terminal metal binding site (in particular, metal binding
residues H6, H13, and H14) is insufficient for such a function.
Predictably, the addition of the phenol group (shown in AQP1,
AQP2, and AQP4) shifted the preferred interaction toward the
more nonpolar and aromatic residues. Of these three
compounds, only AQP4 demonstrated any interaction with
N-terminal residues (E11; Figure S4d). Additionally, only
AQP1 was observed to effectively modulate Cu(II)−Aβ40
aggregation, which is likely associated with the relatively high
affinity of AQP1 for Cu(II) (vide infra). Combined, these
results further imply that antiamyloidogenic activity is not as
much a function of simply where on the monomer a compound
binds but rather a function of a compound’s interaction site(s)
and its ability to interact with other components of the system.
The addition of the 4-(dimethylamino)phenol, instead of the
phenol group, had a slightly unexpected result. While the added
aromaticity promoted interactions of AQDA1−3 with the
central self-recognition sequence (like AQP1 and AQP2), it
also maintained contacts with more N-terminal residues (E11
and V12) by AQDA1 and AQDA2 which were similar to the
interactions observed in both the nonreactive AQ1 and AQ2,
along with the functional parent, ML (Figure 5 and Figure S4).
In addition, like ML, it is possible that having the slightly polar
dimethylamino moiety on the framework appended to the
added hydrophobicity of the phenol group remediates the shift
toward hydrophobic residues seen for the AQP compounds.
The dimethylamino functionality is indicated to be a moiety
that is able to tune the interaction of hydrophobic compounds
toward more polar peptide regions, functioning as a chemical
rheostat. It is also coupled with consistent reactivity against Aβ.
Thus, having modest hydrophilicity (in this case, instilled by
the dimethylamino functionality) may promote the antiamy-
loidogenic activity of the compounds, as observed in both ML
and the AQDA derivatives.
(AQDA1−3) were able to disassemble preformed metal-free
Aβ40/Aβ42 and metal−Aβ40/Aβ42 aggregates (Figures S1b,
and S2b, third column, lanes 1−3). Consistent with the
observations from the Cu(II)−Aβ40 inhibition experiment,
AQP1 also presented an ability to generate a distribution of
smaller MW species only under Aβ40 conditions; however, the
bands appeared at a higher MW region (i.e., 100−260 kDa),
relative to the compounds containing the 4-(dimethylamino)-
phenol moiety which produced a more disperse MW range of
aggregates (i.e., ca. 4−260 kDa). Overall, our in vitro gel/
Western blot and TEM studies suggest that the 4-
(
dimethylamino)phenol functionality as a critical moiety for
the antiamyloidogenic properties of ML.
Direct Interaction between Soluble Metal-Free Aβ
and AQ Derivatives. In order to elucidate the potential
binding regions between Aβ40 and the AQ derivatives, 2D
band-selective optimized flip-angle short transient heteronu-
clear multiple quantum coherence (SOFAST-HMQC) NMR
2
3,31
was employed.
Chemical shift perturbation (CSP) is
indicative of an altered electronic environment around the
assigned residue which is likely the result of interaction with the
derivatives (Figure 5 and Figure S4). Despite the various
chemical alterations to the ML framework investigated herein,
there are two primary structural regions of Aβ that show
interaction with the AQ derivatives observed by NMR.
Compounds altered either the N-terminal residues near the
metal binding site of Aβ40 (i.e., predominantly E11 and V12)
or the central hydrophobic residues within the self-recognition
7
,8,11,12
sequence (LVFFA).
It was previously demonstrated that
ML predominantly perturbs V12 and Q15, suggesting that the
N-terminal contacts may be partially responsible for its efficacy
23
and desirable for future chemical tools.
Of the AQ derivatives studied by NMR, both AQ1 and AQ2
have CSP profiles most similar to ML (Figure 5 and Figure S4).
These molecules are not shown to have any ML-like
antiamyloidogenic activity, which suggests that simply targeting
The interactions between Aβ and the AQ derivatives
examined by 2D NMR were further visualized and probed by
docking studies that were performed by employing AutoDock
4
6
Vina and the previously reported NMR structure of
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Figure 5. Interactions of AQ1, AQP1, and AQDA1 with monomeric Aβ40, monitored by SOFAST-HMQC NMR. (a−c) SOFAST-HMQC NMR
spectra (zoomed in view from 7.9 to 8.4 ppm; top) and chemical shift perturbations (CSPs) (bottom) of Aβ40 upon treatment with (a) AQDA1,
(
b) AQP1, or (c) AQ1. Two horizontal lines represent the average chemical shift (dashed line) plus one standard deviation (dotted line). Residues
which show no CSP are the result of unresolved peaks in the spectra.
4
3
monomeric Aβ40 (PDB 2LFM) (Figure S5). The docking
results showed that, for most conformations of Aβ, the ligands
bound almost exclusively in the pocket formed by the folding of
the N-terminal random coil and α-helix and their adducts with
the peptide were stabilized by nonspecific and/or direct
intermolecular interactions (e.g., hydrogen bonding, π−π
stacking). These interactions had calculated binding energies
ranging from −7.0 to −4.6 kcal/mol (Figure S5). Similar to the
findings from our 2D NMR experiments, the AQP derivatives
notable and replicable reduction in the total Cu(II)-bound Aβ
species was identified upon incubation with many of the ligands
studied (AQP1, AQDA1, AQDA2, and ML), when compared
to the baseline levels prior to small molecule incubation
supporting their metal chelation activity (Figure S6). In the
absence of a source of Cu(II), no ligand binding was observed
(data not shown).
To gain further insight into the structures of the complexes
between Cu(II)-treated Aβ40 and the AQ derivatives, we
measured and compared the IM arrival time distributions for
these complexes (Figure 6i; see Table S2 for the supporting
collisional cross section (CCS) data). Our results indicate that
AQP1 and AQP4 binding to Cu(II)-treated Aβ leads to a
distinct conformation shift when compared to the compound-
untreated metal-free and metal-bound Aβ states. In particular,
close analysis of the IM drift time presents a slight perturbation
of the Cu(II)−Aβ40 conformations toward more expanded
conformers compared to compound-free Cu(II)-associated
Aβ40. These data contrast with the results for previously
studied small molecules which induced conformational
(
AQP1, AQP2, and AQP4) containing the more hydrophobic
phenol functionality were observed to penetrate more deeply
into the N-terminal pocket and appeared to interact more
tightly with the central self-recognition sequence. Conversely,
AQ1 and AQ2 appeared to dock more toward the N-terminal
residues while the derivatives equipped with the 4-
(
dimethylamino)phenol functionality (AQDA1−3) showed a
tendency to maintain both close interactions with the N-
terminal residues, mostly by hydrogen bonding, while
simultaneously establishing close contacts with the α-helical
central hydrophobic region. Overall, these docking findings
support the 2D NMR investigations that identified the aptitude
of the dimethylamino functionality to tune the interaction of
hydrophobic compounds toward the N-terminal hydrophilic
residues.
31,32,35
compaction upon coincubation with Aβ.
Such a differ-
ence in the conformations generated upon AQP1 treatment
(i.e., the more expanded structures compared to the
31,32,35
compaction observed previously)
may suggest that
Analysis of AQ Derivatives Incubated with Aβ40 by
Ion Mobility−Mass Spectrometry. To further explore the
interactions between Aβ40 and the AQ derivatives studied
herein, we applied nanoelectrospray−ionization mass spec-
trometry (nESI−MS) combined with ion mobility−mass
spectrometry (IM−MS), optimized for the detection of
AQP1’s ability to alter the Aβ40 aggregation pathway in the
presence of Cu(II) may be directed mainly by its metal
chelation properties rather than its induction of structural
alteration of Aβ. A metal chelation dependence would also
further validate why AQP4 [the compound which binds Cu(II)
much weaker than AQP1 (vide infra)] does not modulate the
aggregation of copper-bound Aβ40 in vitro.
33,34
noncovalent protein complexes.
The MS data presented
in Figure 6 highlight that, among the multifunctional
derivatives, only AQP1 and AQP4 were capable of binding
Cu(II)-treated Aβ40. While no other small molecules were
observed to form complexes with Cu(II)-associated Aβ40, a
On the basis of the absence of any observable Aβ−ligand
complexes for the AQ derivatives equipped with the 4-
(dimethylamino)phenol functionality (i.e., AQDA1, AQDA2,
AQDA3), two mechanisms are proposed to rationalize the
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Figure 6. MS and IM−MS data for AQ1, AQ4, AQP1, AQP4, AQDA1−3, and ML upon addition of CuCl . MS spectra of (a) AQ1, (b) AQ4, (c)
2
AQDA1, (d) AQDA2, (e) AQDA3, (f) ML, (g) AQP1, and (h) AQP4. (i) IM-MS drift time analysis. Collision cross section data for all ion mobility
data sets are presented in Table S2. L = ligand (i.e., AQ1, AQ4, AQDA1−3, ML, AQP1, AQP4). Asterisk indicates a contaminant refractory to our
purification methods.
activities of these molecules as Aβ modulators. First, these
remaining small molecules may target larger, higher-order
oligomers that are too transient for IM−MS detection under
the conditions used herein. While the analyses of the
interactions between small molecules, such as these, and Aβ
dimers are technically possible, the presence of Cu-based salt
cluster chemical noise has prevented this analysis. Second,
AQDA1, AQDA2, and ML are shown to sequester Cu(II) from
Aβ, and thus our data suggests the contribution of the ligands’
metal chelation properties toward its control of metal-induced
Aβ aggregation.
the CuCl2 experiments, the ZnCl2 binding peaks for the
derivatives containing the 4-(dimethylamino)phenol function-
ality were relatively red shifted compared to the AQP
derivatives (i.e., new peaks growing in at ca. 350 and 450
nm; Figure S8h−j). No significant changes in the UV−vis
spectra could be identified for AQ3 and AQP3 under the
35
1
experimental conditions employed; therefore, H NMR spec-
troscopy was utilized to further probe their Zn(II) binding.
Introduction of 3.5 equiv of ZnCl to a CD CN solution of
2
3
AQ3 induced a slight downfield chemical shift of the quinoline
protons, demonstrating the potential involvement of the
nitrogen donor atoms from the primary amine and quinoline
ring in Zn(II) coordination (Figure S9). No significant
Metal Binding Properties of AQ Derivatives. UV−
1
visible (UV−vis) and H NMR spectroscopy were first
employed in order to probe the effects of structural
modifications on the metal binding properties of the multi-
functional derivatives. Upon coincubation of the ligands with
increasing amounts of CuCl or ZnCl (Figures S7 and S8),
chemical shifts were observed when ZnCl was added to a
2
solution of AQP3; however, this is most likely a result of its
limited solubility under the experimental conditions. Overall,
our UV−vis and NMR studies indicate the ability of the
structural derivatives of ML to bind both Cu(II) and Zn(II).
In order to comprehend the solution speciation of the AQ
derivatives in the absence and presence of Cu(II) and attempt
to determine the effects of structural variations on the Cu(II)
binding affinity of our ligands, UV−vis variable-pH titration
experiments were conducted. First, spectrophotometric titra-
tions of the ligands (i.e., AQP1, AQP4, AQDA1−3) were used
2
2
new optical bands and/or changes in the absorbance intensity
were observed. Decreases in the absorbance of the peaks at ca.
2
50/340 (for AQ1) and 250/400 nm (for AQ3) followed by
the growth of new bands at ca. 300 and/or 450 nm (for AQ1−
) were discernible as CuCl was titrated into solution (Figure
3
2
S7a−c). AQ derivatives augmented with phenols (i.e., AQP1−
) produced new peaks at ca. 320 and 430 nm in the presence
of CuCl (Figure S7d−g), while the compounds with the 4-
4
to estimate the acidity constants (pK ) [see Figure S10; for
2
a
(
dimethylamino)phenol moiety (i.e., AQDA1−3) generated
AQP1, pKa2 = 3.67(4), pKa3 = 9.92(6); for AQP4, pKa2
=
new optical bands at ca. 380 and 470 nm (Figure S7h−j).
3.78(8), pK = 10.11(8); for AQDA1, pK = 3.72(9), pK =
a3
a1
a2
Administration of ZnCl into solutions of AQ derivatives
6.61(5), pKa3 = 8.99(6); for AQDA2, pKa1 = 3.21(9), pKa2
4.82(6), pKa3 = 7.69(4); for AQDA3, pKa1 = 2.30(8), pKa2
=
=
2
resulted in less noticeable spectral changes when compared to
the CuCl results (Figure S8). Optical shifts from ca. 350 to
3.82(3), pKa3 = 6.73(5)]. The solution speciation diagrams
depict the presence of three species for the phenol derivatives,
AQP1 and AQP4 (anionic, neutral, and monoprotonated
2
3
00 nm were detected upon increased addition of ZnCl to
2
solutions of AQ1 and AQ2 (Figure S8a,b). Incubation of
AQP1, AQP2, and AQP4 with ZnCl caused various spectral
species; LH , L, and LH), and predict the neutral ligand (L)
2
−1
changes at ca. 250, 300, and 350 nm (Figure S8d,e,g). Similar to
predominantly being present at physiological pH (i.e., 7.4;
G
DOI: 10.1021/acs.inorgchem.6b00525
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Inorganic Chemistry
Article
Figure 7. Solution speciation studies of AQP1, AQP4, and AQDA1−3 in the presence of Cu(II). UV−vis variable-pH titration spectra (left) and
solution speciation diagrams (right) of (a) AQP1, (b) AQP4, (c) AQDA1, (d) AQDA2, and (e) AQDA3 upon incubation with Cu(II) (F
=
Cu
fraction of species at given pH). Stability constants (logβ) of Cu(II)−L complexes (L = AQP1, AQP4, and AQDA1−3) are summarized in the table.
a
Charges are omitted for clarity. The error in the last digit is shown in parentheses.
Figure S10a,b). Due to the protonation of the dimethylamino
in the R position, presented the strongest affinity for Cu(II)
2
group, AQDA derivatives contain an additional diprotonated
compared to the other tetradentate ligands measured herein
(i.e., AQP4, AQDA3), thus possibly explaining the ability of
AQP1 to modulate the aggregation of Cu(II)−Aβ40 (Figure 2
and Figure S1). This trend may be explained by the ability of
AQDA1 and AQP1 to form 1:2 complexes [Cu(II):ligand] in
addition to the 1:1 stoichiometry observed for the other
derivatives (Figure 7a,c). Comparison of the apparent Kd
values of AQDA1 with AQP1 and ML with AQP4 indicates
that the dimethylamino functionality slightly increases the
metal binding affinity, as would be expected with the
installation of a electron donating group located para to the
oxygen donor atom of the phenol. AQDA2 and AQDA3 were
species (LH ) in the pH range examined and exist in a mixture
2
of neutral and cationic (for AQDA1) or anionic and neutral
(
for AQDA2 and AQDA3) species (Figure S10c−e). Overall,
the relative abundance of the neutral form of the ligands at pH
7
.4 (ca. 100% for AQP1 and AQP4; ca. 50% for AQDA1−3)
27
may explain their potential BBB permeability, as suggested by
Lipinski’s rules, the PAMPA-BBB assay, and calculated logBB
values (Table 1 and Table S1).
Once pK values were obtained for the ligands of interest,
a
solution speciation experiments in the presence of CuCl were
2
carried out to determine if structural modifications to ML’s
framework at sites 2 and 3 could alter its high apparent
determined to have larger K values [lower binding affinity for
d
dissociation constant for Cu(II) (picomolar K at pH 7.4),
Cu(II)] than ML. The slightly smaller K of AQDA3 for
d
d
while still maintaining competitive metal binding with Aβ (e.g.,
Cu(II), compared to AQDA2, is most likely due to the weak
contribution from the ester functionality. Overall, tuning metal
binding strengths of the multifunctional derivatives is able to be
accomplished through structural modifications at the R and R
2
7
,9,11,12,23
nanomolar range).
constants (logβ) and values of pCu (pCu ≈ −log[Cuunchelated])
Figure 7), the approximate dissociation constants (K
On the basis of the stability
(
=
d
1
[
Cuunchelated]) for the multifunctional derivatives were deter-
positions.
mined. As summarized in the table in Figure 7, the more inert
Cu(II)−ligand complexes were shown in the order of AQDA1,
AQP1, AQP4, AQDA3, and AQDA2. Interestingly, the
tridentate ligands, AQDA1 and AQP1, with hydrogen atoms
Competition reactions, monitored by UV−vis, were also
conducted to examine the selectivity of AQP4 and AQDA1−3
for Cu(II) over other biologically relevant divalent metal ions
(i.e., Mg(II), Ca(II), Mn(II), Fe(II), Co(II), Ni(II), Zn(II)). As
H
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Inorganic Chemistry
Article
shown in Figure S11, in the presence of equimolar (Figure
S11a) or excess metal ions (20 equiv; Figure S11b), the ligands
still exhibited spectral changes consistent with the formation of
Cu(II) complexes. Fe(II) did appear to competitively interact
with AQP4, AQDA1, and AQDA2, especially at excess
concentrations. It is also worth noting that the exact
quantification of the selectivity for Cu(II) over Ni(II) and
Zn(II) for AQP4 and Co(II) for AQDA1 could not be
determined due to the optical overlap of their respective metal
binding bands, but the overall spectral changes were suggestive
of preferential binding to Cu(II) (Figure S11). Collectively,
these results present that AQ derivatives can competitively bind
to Cu(II) over other biologically available divalent metal ions
and that their metal affinities can be modulated through
structural modifications.
In order to evaluate the antioxidant capacity of the AQ
derivatives, the Trolox equivalent antioxidant capacity (TEAC)
assay which measures the ability of the multifunctional
derivatives to quench preformed ABTS cation radicals
•
+
(ABTS ; ABTS = 2,2′-azino-bis(3-ethylbenzothiazoline-6-
15,37
sulfonic acid)
was performed using lysates of human
neuroblastoma SK-N-BE(2)-M17 cells. Cell lysates were
utilized in order to get a more accurate measure of the
antioxidant capacity of the AQ derivatives in a more biologically
relevant heterogeneous environment. As shown in Figure 8b,
the compounds containing the phenol or 4-(dimethylamino)-
phenol functionalities were able to scavenge free radicals about
two times more effectively than that of the water-soluble
vitamin E analogue, Trolox. These findings are in-line with the
38−40
known antioxidant properties of phenols.
The most
Biological Properties: ROS Formation Control, Free
Radical Scavenging Capacity, and Cytotoxicity. The
extent to which structural modifications affect the biological
properties of our multifunctional derivatives was also
investigated. The ability to control the redox cycling between
Cu(I) and Cu(II) in order to reduce ROS production through
Fenton-like chemistry was first analyzed by the 2-deoxyribose
assay which measures the capacity of ligands to control the
efficient antioxidants, AQDA1 and AQDA2, were still less
active than ML which was determined to be ca. 2.5 times as
23
effective as Trolox, indicating that the primary alcohol in the
R2 position may contribute to the antioxidant activity of ML. In
addition, an ester at the R site appeared to reduce both the
2
abilities of the compounds to control ROS generation and
scavenge free radicals relative to those of the other multifunc-
tional derivatives (Figure 8a,b).
2
3,36
formation of copper-catalyzed hydroxyl radicals.
As
Finally, the MTT assay [MTT = 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] was employed
to evaluate the toxicity of the AQ derivatives in the mouse
Neuro-2a (N2a) neuroblastoma cell line with and without
CuCl and ZnCl (Figures S12). Cell viability of ca. 80% was
depicted in Figure 8a, copper-mediated generation of hydroxyl
2
2
measured for N2a cells treated with 5 μM of the AQ derivatives
in the absence and presence of metal ions (CuCl or ZnCl )
2
2
(
Figure S12a,b). A little more fluctuation in cell viability was
observed upon increasing the concentration of compound to 10
μM with and without metal ions, but still most derivatives
appeared to retain values around ca. 75−85% (Figure S12c,d).
In particular, with the exception of ML, the multifunctional
derivatives containing the 4-(dimethylamino)phenol function-
ality are indicated to be relatively more cytotoxic. Overall, our
cell studies suggest that the structural variations in the
framework may also trigger its differing levels of toxicity in
living cells.
Figure 8. Biological activities of small molecules. (a) Inhibitory activity
of AQ1, AQ3, AQ4, AQDA1, and AQDA3 toward Cu-mediated ROS
formation as determined by the 2-deoxyribose assay. The absorbance
values are normalized to the ligand-free condition ([CuCl ] = 10 μM;
2
[
ligand] = 125 μM). (b) Antioxidant activity of AQ1−3, AQP1,
AQP2, AQP4, and AQDA1−3, identified by the TEAC assay using
cell lysates. The TEAC values are relative to that of the vitamin E
analogue, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid).
CONCLUSION
■
A series of derivatives, AQ1−4, AQP1−4, and AQDA1−3, was
developed based on the structural framework of the multifunc-
tional ligand, ML, in order to tune its affinity for Cu(II) and to
establish a structure−reactivity understanding of ML’s activities
to modulate metal-free and metal-bound Aβ aggregation,
control metal-mediated ROS formation, and scavenge free
radicals. Only compounds augmented with the dimethylamino
functionality displayed noticeable modulation of both metal-
free and metal-treated Aβ aggregation in vitro, with the
exception of AQP1 which exhibited its ability to regulate the
aggregation of Cu(II)−Aβ40, which was most likely aided by its
metal chelating properties (i.e., pCu = 11.44 at pH 7.4). On the
basis of NMR investigations, this dimethylamino moiety
appears to act to control the distribution of the compounds’
interaction with the N-terminal metal binding region and/or
the central self-recognition sequence of Aβ40. ML was
previously found to preferentially target the polar N-terminal
residues, which suggests that the dimethylamino group is at
radicals was most significantly reduced upon treatment with
AQDA1. Relative to AQDA1, the other derivatives evaluated
showed very little aptitude to attenuate hydroxyl radical
formation, possibly due to their metal binding properties,
including weaker binding affinity for Cu(II) compared that of
AQDA1 or ML. Still, ML appeared to be about twice as
efficient at controlling the formation of hydroxyl radicals
relative to AQDA1 (A/A of ca. 0.40 for AQDA1; A/A of ca.
0
0
2
3
0
.20 for ML). The enhanced ROS formation control of ML is
most likely due to its tetradentate metal binding center which
can easily accommodate the preferred square planar geometry
of Cu(II) but prohibit the generation of linear or tetrahedral
geometries favored by Cu(I). In fact, on the basis of
crystallographic data reported for AQP1, AQDA1 may be
able to facilitate a tetrahedral copper-binding mode depending
on the metal-to-ligand stoichiometry and the pH of the
23
least partly responsible for Aβ interaction. Favoring these
interactions may be important for directing the formation of
stable ternary complexes of Aβ−metal−ligand. Under our MS
2
7
solution.
I
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Inorganic Chemistry
Article
2
3
and IM−MS conditions, only noncovalent interactions between
Aβ and AQP1 or AQP4 when Cu(II) was present with slight
structural elongation were observed, rather than compaction of
the peptide which has been previously reported for other
quinolin-8-yl)amino)methyl)phenol (ML) were prepared by adapt-
ing previously reported methods.
2
-(((2-Methylquinolin-8-yl)amino)methyl)phenol (AQP2). To
a solution of dry ethyl acetate (16 mL) was added 2-methylquinolin-8-
amine (AQ2) (300 mg, 1.89 mmol). Salicylaldehyde (198 μL, 1.89
mmol) was slowly added to the reaction mixture. The reaction mixture
was then protected from the light and allowed to stir overnight for 24
h. After 24 h, the reaction mixture was concentrated and dried. To a
solution of dichloroethane (DCE) (16 mL) was added sodium
triacetoxyborohydride (804 mg, 3.78 mmol). The sodium triacetox-
yborohydride solution was then slowly added to the dried reaction
mixture and allowed to stir for 48 h (protected from the light). After
3
1,32,35
inhibitors.
Preferential transient interactions with higher-
order oligomers that cannot easily be detected by IM−MS may
explain the absence of complexation peaks in the AQDA1−3-
treated samples that most noticeably perturbed metal-free and
metal-treated Aβ aggregation in vitro. Structural modifications
to ML also had drastic effects on its metal binding properties,
ROS formation control, and free radical scavenging capacity.
Substitution of the primary alcohol on ML with a hydrogen
atom allowed for the formation of 1:1 and 1:2 (metal:ligand)
complexes, providing a higher binding affinity for Cu(II) with
respect to the other AQ derivatives. Structural variations also
had impacts on compounds’ abilities to control ROS formation
and scavenge free radicals. AQDA1, with a hydrogen atom at
4
8 h, the reaction mixture was concentrated and purified by column
chromatography (Al O , 1:10 ethyl acetate (EtOAc)/hexanes (Hx), R
f
0.23) to yield the final product (light yellow powder, 241 mg, 0.912
2
3
=
1
mmol, 48%). H NMR (400 MHz, (CD ) SO)/δ (ppm): 2.62 (3H, s),
3
2
4
(
.41 (2H, d, J = 4.0 Hz), 6.57 (1H, d, J = 8.0 Hz), 6.68 (2H, m), 6.81
1H, d, J = 8.0 Hz), 6.96 (1H, d, J = 8.0 Hz), 7.03 (1H, t, J = 8.0 Hz),
7.18 (2H, m), 7.34 (1H, d, J = 8.0 Hz), 8.05 (1H, d, J = 8.0), 9.07 (1H,
s). 13C NMR (100 MHz, (CD
SO)/δ (ppm): 25.3, 42.1, 105.2,
13.6, 115.4, 119.2, 122.7, 125.7, 126.7, 127.1, 128.2, 136.6, 137.3,
the R site, was indicated to be the most efficient at controlling
)
3 2
2
1
1
2
the generation of hydroxyl radicals, while the compounds
containing the phenol or 4-(dimethylamino)phenol groups
+
44.2, 155.6, 155.7. HRMS Calcd for C H N O [M + H] ,
17 17
2
65.1341; found 256.1331.
Methyl 8-((2-hydroxybenzyl)amino)quinoline-2-carboxylate
AQP3). A solution of methyl 8-aminoquinoline-2-carboxylate (AQ3)
100 mg, 0.494 mmol) was utilized to prepare AQP3 following a
procedure identical to the one described for AQP2. AQP3 was
purified by column chromatography (SiO , 1:5 EtOAc/dichloro-
(AQP1−4 or AQDA1−3 and ML) were much more potent
antioxidants with respect to their AQ counterparts (AQ1−4).
Conversely, the endowment with an ester, shown in AQ3,
AQP3, or AQDA3, appeared to negatively alter the capacity to
inhibit ROS generation and scavenge free radicals. Overall, by
employing a series of AQ derivatives, a relationship between
structures of the small molecules and reactivities toward targets
found in AD (e.g., metal-free Aβ/metal−Aβ, metals, ROS) was
established. These structure−reactivity insights, gleaned
through our studies, may aid in further design of more
sophisticated multifunctional ligands, especially once the degree
of transferability of these studies has been determined.
(
(
2
methane (DCM), R
powder, 79.3 mg, 0.257 mmol, 52%). H NMR (400 MHz,
CD ) SO)/δ (ppm): 3.91 (3H, s), 4.46 (2H, s), 6.67−6.70 (2H,
= 0.72) to yield the final product (yellow-orange
f
1
(
3
2
m), 6.82 (1H, d, J = 8.0 Hz), 7.02 (1H, t, J = 8.0 Hz), 7.07 (1H, d, J =
8
.0 Hz), 7.19 (1H, d, J = 8.0 Hz), 7.41 (1H, t, J = 8.0 Hz), 8.04 (1H, d,
J = 12.0 Hz), 8.34 (1H, d, J = 12.0 Hz), 9.66 (1H, s). C NMR (100
MHz, (CD ) SO)/δ (ppm): 40.3, 53.0, 105.8, 113.2, 115.5, 119.3,
21.6, 125.2, 128.4, 129.0, 130.8, 137.1, 137.7, 144.3, 145.5, 155.7,
65.6. HRMS Calcd for C H N O [M + H] , 309.1239; found
09.1231
-(((2-(Hydroxymethyl)quinolin-8-yl)amino)methyl)phenol
AQP4). A solution of (8-aminoquinolin-2-yl)methanol (AQ4) (100
mg, 0.574 mmol) was utilized to prepare AQP4 following an identical
procedure as the one described for AQP2. AQP4 was purified by
column chromatography (SiO , 1:1 EtOAc/Hx, R = 0.50) to yield the
final product (light brown powder, 64.8 mg, 0.231 mmol, 40%). H
NMR (400 MHz, (CD ) SO)/δ (ppm): 4.41 (2H, d, J = 8.0 Hz), 4.70
(2H, d, J = 8.0 Hz), 5.45 (1H, t, J = 8.0 Hz), 6.55 (1H, d, J = 8.0 Hz),
6.67 (1H, t, J = 8.0 Hz), 6.81 (1H, d, J = 8.0 Hz), 6.92 (1H, t, J = 8.0
Hz), 6.95−7.04 (2H, m), 7.16 (1H, d, J = 8.0 Hz), 7.22 (1H, t, J = 8.0
Hz), 7.53 (1H, d, J = 8.0 Hz), 8.15 (1H, d, J = 12.0 Hz), 9.56 (1H, s).
13
3
2
1
1
3
+
18
17
2
3
EXPERIMENTAL SECTION
■
2
Materials and Methods. All reagents were purchased from
commercial suppliers and used as received unless otherwise noted.
Aβ40 and Aβ42 were purchased from Anaspec (Aβ42 =
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA;
Fremont, CA). NMR and mass spectrometric analysis of small
molecules were conducted on a 400 MHz Varian NMR spectrometer
and a Micromass LCT electrospray time-of-flight (TOF) mass
spectrometer, respectively. Trace metal contamination was removed
from buffers and solutions used for metal binding and Aβ experiments
(
2
f
1
3
2
(vide infra) by treating with Chelex overnight (Sigma-Aldrich, St.
13
Louis, MO). Optical spectra were recorded on an Agilent 8453 UV−
visible (UV−vis) spectrophotometer. TEM images were taken using a
JEOL JEM-2100 transmission electron microscope (UNIST Central
Research Facilities, Ulsan National Institute of Science and
Technology, Ulsan, Republic of Korea). Absorbance values for
biological assays, including cell viability assay, PAMPA-BBB, 2-
deoxyribose assay, and TEAC assay, were measured on a Molecular
Devices SpectraMax 190 microplate reader (Sunnyvale, CA). Ion
mobility-mass spectrometry experiments investigating the interaction
of AQ derivatives with Aβ in the absence and presence of Cu(II) were
acquired using a quadrupole-ion mobility-TOF (Q-IM-TOF) mass
spectrometer (Waters Synapt G2, Milford, MA) equipped with a
nanoelectrospray ionization (nESI) source. NMR studies of small
molecules with Aβ were performed on a 600 MHz Bruker NMR
spectrometer (University of Michigan, Ann Arbor, MI).
C NMR (100 MHz, (CD ) SO)/δ (ppm): 41.9, 65.2, 105.2, 113.5,
15.4, 119.2, 119.6, 125.7, 127.5, 127.7, 128.1, 136.7, 136.9, 144.6,
3 2
1
1
+
55.6, 158.9. HRMS Calcd for C H N O [M + H] , 281.1290;
17
17
2
2
found 281.1280.
4
-(Dimethylamino)-2-((quinolin-8-ylamino)methyl)phenol
(AQDA1). To a solution of dry EtOAc (5.0 mL) was added quinolin-
8-amine (AQ1) (100 mg, 0.694 mmol). 5-(Dimethylamino)-2-
hydroxybenzaldehyde (DHB) (115 mg, 0.694 mmol) was slowly
added to the reaction mixture. The reaction mixture was then
protected from the light and allowed to stir overnight for 24 h. After
24 h, the reaction mixture was concentrated and dried. To a solution
of DCE (5.0 mL) was added sodium triacetoxyborohydride (212 mg,
1.39 mmol). The sodium triacetoxyborohydride solution was then
slowly added to the dried reaction mixture and allowed to stir for 24 h
(protected from the light). After 24 h, the reaction mixture was
concentrated and dissolved in dry methanol. Sodium borohydride
(150 mg, 3.97 mmol) was added to the reaction mixture at 0 °C and
allowed to stir for 2 h. After 2 h, the reaction mixture was quenched
Syntheses. The compounds, quinolin-8-amine (AQ1) and 2-
methylquinolin-8-amine (AQ2), were purchased from TCI chemicals.
2
8
5
-(Dimethylamino)-2-hydroxybenzaldehyde (DHB), methyl 8-ami-
29,31
noquinoline-2-carboxylate (AQ3),
methanol (AQ4),
(8-aminoquinolin-2-yl)-
2-((quinolin-8-ylamino)methyl)phenol
and 4-(dimethylamino)-2-(((2-(hydroxymethyl)-
with sodium bicarbonate and H O, extracted with DCM (3×), and
purified by column chromatography (SiO , 1:1 EtOAc/Hx, R = 0.43).
The solid was recrystallized in DCM/Hx to afford the final product
2
2
9,31
2
f
2
9,30
(
AQP1),
J
DOI: 10.1021/acs.inorgchem.6b00525
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Inorganic Chemistry
Article
1
(
light brown powder, 140 mg, 0.479 mmol, 69%). H NMR (400
compound were incubated at 37 °C with constant agitation for 4, 8,
and 24 h.
MHz, (CD ) SO)/δ (ppm): 2.63 (6H, s), 4.36 (2H, d, J = 4.0 Hz),
3
2
6
.49 (1H, dd, J = 8.0 Hz, J = 4.0 Hz), 6.66−6.69 (2H, m), 6.74 (1H, d,
J = 4.0 Hz), 6.79 (1H, t, J = 8.0 Hz), 7.01 (1H, d, J = 8.0 Hz), 7.29
1H, t, J = 8.0 Hz), 7.46 (1H, dd, J = 8.0 Hz, J = 4.0 Hz), 8.16 (1H, dd,
Gel Electrophoresis with Western Blotting. The samples from
the inhibition and disaggregation experiments were analyzed by gel
electrophoresis with Western blot using an anti-Aβ antibody
(
2
3,31,32,35
J = 6.8 Hz, J = 8.4 Hz), 8.69 (1H, dd, J = 2.4 Hz, J = 4.0 Hz), 8.83 (1H,
(6E10).
Each sample (10 μL) was separated on a 10−20%
s). 13C NMR (100 MHz, (CD ) SO)/δ (ppm): 41.8, 42.7, 105.2,
Tris-tricine gel (Invitrogen, Grand Island, NY). Following separation,
the proteins were transferred onto nitrocellulose, which was blocked
with bovine serum albumin (BSA, 3% w/v, RMBIO, Missoula, MT) in
Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 4 h
at room temperature. The membranes were incubated with antibody
(6E10, 1:2000, Covance, Princeton, NJ) in a solution of 2% BSA (w/v
in TBS-T) overnight at 4 °C. After washing, the horseradish
peroxidase-conjugated goat antimouse secondary antibody (1:5000)
in 2% BSA was added for 1 h at room temperature. ThermoScientific
SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific, Rockford, IL), Biosesang ECL Plus kit (Biosesang,
3
2
1
1
3
13.6, 113.7, 115.4, 116.0, 122.1, 125.9, 128.2, 128.7, 136.4, 138.0,
+
44.8 145.0, 147.3, 147.6. HRMS Calcd for C H N NaO [M + Na] ,
18
19
3
16.1426; found 316.1418.
-(Dimethylamino)-2-(((2-methylquinolin-8-yl)amino)-
methyl)phenol (AQDA2). A solution of 2-methylquinolin-8-amine
AQ2) (100 mg, 0.632 mmol) was utilized to prepare AQDA2
4
(
following an identical procedure as the one described for AQDA1.
AQDA2 was purified by column chromatography (SiO , 1:3 EtOAc/
Hx, R = 0.33). The solid was recrystallized in EtOAc/Hx to afford the
final product (light brown powder, 83.5 mg, 0.272 mmol, 43%). H
NMR (400 MHz, (CD ) SO)/δ (ppm): 2.63 (3H, s), 2.68 (6H, s),
4
2
f
1
41
Gyeonggi-do, Republic of Korea), or a homemade ECL kit was
used to visualize the results on a ChemiDoc MP Imaging System (Bio-
Rad, Hercules, CA).
3
2
.38 (2H, d, J = 8.0 Hz), 6.53 (1H, dd, J = 4.0 Hz, J = 8.0 Hz), 6.64
(
(
8
1H, t, J = 8.0 Hz), 6.68−6.73 (2H, m), 6.77 (1H, d, J = 8.0 Hz), 7.00
Transmission Electron Microscopy (TEM). Samples for TEM
1H, d, J = 8.0 Hz), 7.25 (1H, t, J = 8.0 Hz), 7.36 (1H, d, J = 8.0 Hz),
23,31,32,35
.07 (1H, d, J = 8.0 Hz), 8.86 (1H, s). 1 C NMR (100 MHz,
3
were prepared according to previously reported methods.
Glow-discharged grids (Formar/Carbon 300-mesh, Electron Micros-
copy Sciences, Hatfield, PA) were treated with Aβ samples from the
inhibition and disaggregation experiments (5 μL) for 2 min at room
temperature. Excess sample was removed using filter paper followed by
(
CD ) SO)/δ (ppm): 25.3, 41.9, 43.0, 105.4, 113.6, 113.7, 115.4,
3 2
1
1
3
16.0, 122.6, 125.9, 126.7, 127.1, 136.6, 137.4, 144.5, 144.8, 147.6,
+
55.6. HRMS Calcd for C H N O [M + H] , 308.1763; found
19
22
3
08.1762.
Methyl 8-((5-(dimethylamino)-2-hydroxybenzyl)amino)-
quinoline-2-carboxylate (AQDA3). To a solution of dry EtOAc
washing three times with ddH O. Each grid was incubated with uranyl
2
acetate (1% w/v ddH O, 5 μL, 1 min). Upon removal of excess uranyl
2
acetate with filter paper, the grids were dried for at least 30 min at
room temperature before measurement. Images from each sample
were taken on a JEOL JEM-2100 transmission electron microscope
(15 mL) was added methyl 8-aminoquinoline-2-carboxylate (AQ3)
(100 mg, 0.494 mmol). 5-(Dimethylamino)-2-hydroxybenzaldehyde
(DHB) (82.6 mg, 0.494 mmol) was slowly added to the reaction
(
UNIST Central Research Facilities, Ulsan National Institute of
Science and Technology, Ulsan, Republic of Korea) at 120 kV and
5 000× magnification.
D NMR Experiments. The interactions of Aβ40 monomer with
mixture. The reaction mixture was then protected from the light and
allowed to stir overnight for 24 h. After 24 h, the reaction mixture was
concentrated and dried. To a solution of DCE (15 mL) was added
sodium triacetoxyborohydride (209 mg, 0.988 mmol). The sodium
triacetoxyborohydride solution was then slowly added to the dried
reaction mixture and allowed to stir for 24 h (protected from the
light). After 24 h, the reaction mixture was concentrated and purified
by column chromatography (SiO , 1:1 EtOAc/Hx, R = 0.16). The
2
2
AQ derivatives were interrogated by 2D band-selective optimized flip-
angle short transient heteronuclear multiple quantum coherence
42
15
(
(
SOFAST-HMQC) NMR at 10 °C. Uniformly N-labeled Aβ40
rPeptide, Bogart, GA) was dissolved in 1% NH OH and lyophilized
4
2
f
to ensure the absence of preformed aggregates. The peptide was
solid was recrystallized in EtOAc/Hx to afford the final product
redissolved in 3 μL of DMSO-d (Cambridge Isotope, Tewksbury,
1
6
(
yellow powder, 111 mg, 0.316 mmol, 64%). H NMR (400 MHz,
MA) and diluted by buffer to a final peptide concentration of 80 μM
(
CD ) SO)/δ (ppm): 2.69 (6H, s), 3.99 (3H, s), 4.43 (2H, d, J = 4.0
3
2
(20 mM PO , pH 7.4, 5 mM NaCl, 7% v/v D O). Each spectrum was
4
2
Hz), 6.55 (1H, dd, J = 9.0 Hz, J = 3.0 Hz), 6.71−6.82 (4H, m), 7.13
1H, d, J = 8.0 Hz), 7.47 (1H, t, J = 8.0 Hz), 8.06 (1H, d, J = 8.0 Hz),
.38 (1H, d, J = 9.0 Hz), 8.92 (1H, s). 13C NMR (100 MHz,
CD ) SO)/δ (ppm): 41.4, 42.4, 52.6, 105.6, 112.8, 113.4, 114.9,
obtained using 64 complex t points and a 0.1 s recycle delay on a
1
(
8
(
1
Bruker Avance 600 MHz spectrometer equipped with a cryoprobe.
The data were processed using TOPSPIN 2.1 (Bruker), and
assignment was performed using SPARKY 3.1134 using published
3
2
4
3−45
15.7, 121.1, 124.9, 129.6, 130.4, 136.7, 137.2, 143.8, 144.3, 145.3,
assignments as a guide.
Chemical shift perturbation (CSP) was
+
1
47.2, 165.2. HRMS Calcd for C H N O [M + H] , 352.1661;
20 22 3 3
calculated using the following equation:
found 352.1658.
2
Aβ Aggregation Experiments. All experiments were performed
⎛
⎜
⎝
Δδ ⎞
23,31,32,35
2
N
ΔδNH = ΔδH +
⎟
according to previously published methods.
ments, Aβ40 or Aβ42 was dissolved in ammonium hydroxide
NH OH, 1% v/v, aq), aliquoted, lyophilized overnight, and stored
Prior to experi-
5 ⎠
(
4
Docking Studies. Flexible ligand docking studies for AQ
derivatives against the Aβ40 monomer from a previously determined
at −80 °C. For experiments described herein, a stock solution of Aβ
was prepared by dissolving the lyophilized peptide in 1% NH OH (10
μL) and diluting with ddH O. The concentration of the solution was
determined by measuring the absorbance of the solution at 280 nm (ε
43
4
aqueous solution NMR structure (PDB 2LFM) were conducted
46
2
using AutoDock Vina. Ten (10) conformations were selected from
2
1
0 conformations within the Protein Databank (PDB) file (1, 3, 5, 8,
0, 12, 13, 16, 17, and 20). The MMFF94 energy minimization in
−1
−1
−1
−1
=
1450 M cm for Aβ40 and ε = 1490 M cm for Aβ42). The
peptide stock solution was diluted to a final concentration of 25 μM in
the Chelex-treated buffered solution containing HEPES [20 μM; pH
ChemBio3D Ultra 11.0 was used to optimize the ligand structures for
docking studies. The structural files of the AQ derivatives and the
4
7
7.4 (for metal-free and Zn(II) samples) pH 6.6 (for Cu(II) samples)
peptide were generated by AutoDock Tools and imported into PyRx,
23,31,32,35
4
6
and NaCl (150 μM)]. For the inhibition studies,
compound
which were used to run AutoDock Vina. The search space
dimensions were set to contain the entire peptide. The exhaustiveness
for the docking runs was set to 1024. Docked poses of the ligand with
Aβ were visualized using Pymol.
(
final concentration 50 μM, 1% v/v DMSO) was added to the sample
of Aβ (25 μM) in the absence and presence of a metal chloride salt
CuCl or ZnCl ; 25 μM) followed by the incubation at 37 °C with
(
2
2
constant agitation for 4, 8, and 24 h. For the disaggregation studies, Aβ
with and without metal ions was incubated for 24 h at 37 °C with
constant agitation prior to treatment with compound (50 μM). The
resulting samples containing Aβ, a metal chloride salt, and a
Ion Mobility-Mass Spectrometry. All nanoelectrospray ioniza-
tion MS (nESI-MS) combined with ion mobility-mass spectrometry
(IM-MS) experiments were carried out on a Synapt G2 (Waters,
4
8,49
Milford, MA).
Samples were ionized using a nanoelectrospray
K
DOI: 10.1021/acs.inorgchem.6b00525
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
source operated in positive ion mode. MS instrumentation was
operated at a backing pressure of 2.7 mbar and sample cone voltage of
(200 μL of 1% w/v). After quenching, the reactions were heated at
100 °C for 20 min, and then allowed to cool for 5 min prior to
measurement of their absorbance values at 532 nm. Samples without
ligand were prepared as a control. Experiments were preformed in
4
0 V. Aliquots of Aβ40 peptides (final concentration, 20 μM) were
sonicated for 5 s prior to preincubation with or without a source of
Cu(II) (copper(II) acetate, 20 μM) at 37 °C for 10 min. After
preincubation, samples were titrated with or without ligand (AQ1,
AQ4, AQP1, AQP4, AQDA1−3, or ML; final concentrations: 20, 40,
triplicate. Normalized absorbance values (A/A ) were calculated by
0
taking the absorbance (A) and dividing by the absorbance of the
control (A0).
8
0, and 120 μM) and incubated at 37 °C for 30 min prior to analysis.
Solution conditions were 100 mM ammonium acetate (pH 7.5) with
% v/v DMSO. For control purposes, all data are compared to
incubations of Aβ40 peptides with EGCG under the same
Trolox Equivalent Antioxidant Capacity (TEAC) Assay. The
antioxidant activity of AQ1−3, AQP1−2, AQP4, and AQDA1−3 was
determined by the TEAC assay using human neuroblastoma SK-N-
BE(2)-M17 (M17) cell lysates and was conducted according to a
protocol of the antioxidant assay kits purchased from Cayman
Chemical Company (Ann Arbor, MI) with minor modifications. The
cell line purchased from ATCC (Manassas, VA) was maintained in
media containing 1:1 minimum Essential Media (MEM, GIBCO) and
Ham’s F12K Kaighn’s Modification Media (F12K, GIBCO), 10% (v/
v) fetal bovine serum (FBS, GIBCO), 100 U/mL penicillin (GIBCO),
and 100 mg/mL streptomycin (GIBCO). The cells were grown and
1
3
5
conditions. Collision cross-section (CCS) measurements were
externally calibrated using a database of known values in helium,
with values for proteins that bracket the likely CCS and ion mobility
34,50
values of the unknown ions.
CCS values are the mean average of
five replicates with errors reported as the least-squares product. This
least-square analysis combines inherent calibrant error from drift tube
50
measurements (3%), calibration curve error, and twice the replicate
standard deviation error. Determination of the amount of Cu(II)
bound to Aβ40 was calculated using the total ion count extracted from
the peak of interest at its full width half-maximum using methods
maintained at 37 °C in a humidified atmosphere with 5% CO . The
2
cells were seeded in a 6 well plate and grown to approximately 80−
90% confluence. Cell lysates were prepared following a previously
51
54
previously described. All other conditions are consistent with
reported method with modifications. M17 cells were washed once
32
previously published methods.
with cold PBS (pH 7.4, GIBCO) and harvested by gently pipetting off
adherent cells with cold PBS. A cell pellet was generated by
centrifugation (2000 g for 10 min at 4 °C). This cell pellet was
sonicated on ice (5 s pulses five times with 20 s intervals between each
pulse) in 2 mL of cold Assay Buffer [5 mM potassium phosphate (pH
7.4) containing 0.9% NaCl and 0.1% glucose]. The cell lysates were
centrifuged at 10 000 g for 10 min at 4 °C. The supernatant was
removed and stored on ice until use. To a standard sample 96
microplate, 10 μL of the supernatant cell lysates was delivered
followed by addition of compound, metmyoglobin (2.5 μM), ABTS
(165 μM), and H O (82.4 μM) in order. Compound concentration
Metal Binding Experiments. Metal binding properties of AQ1−
1
3
, AQP1−4, and AQDA1−3 were investigated by UV−vis and H
NMR. UV−vis experiments were carried out in acetonitrile (for AQ2
and AQP3) or a Chelex-treated buffered solution containing 20 mM
HEPES, pH 7.4, and 150 mM NaCl. To a solution of ligand, CuCl or
2
ZnCl was titrated up to 10 equiv at room temperature. The solutions
2
were allowed to equilibrate before further addition of CuCl or ZnCl .
2
2
1
Zn(II) binding to AQ3 was probed by H NMR by slowly titrating up
to 3.5 equiv of ZnCl (17.5 mM) at room temperature in CD CN. To
2
3
examine the metal selectivity of AQP4 and AQDA1−3 for Cu(II), 1
2
2
or 20 equiv of MgCl , CaCl , MnCl , FeCl , CoCl , NiCl , and ZnCl
ranges utilized were as follows: Trolox (45, 90, 135, 180, 225, and 330
μM); AQ1, AQ3, AQP4, AQDA1, and AQDA2 (30, 50, 70, 90, 110,
and 135 μM); AQ2 (30, 70, 110, 150, 190, and 255 μM); AQP1 and
AQP2 (20, 40, 60, 80, 100, and 120 μM); AQDA3 (30, 60, 90, 120,
150, and 180 μM). After 5 min incubation at room temperature on a
shaker, absorbance values at 750 nm were recorded. The percent
inhibition was calculated according to the measured absorbance (%
inhibition = (A0 − A)/A , where A is the absorbance of the
2
2
2
2
2
2
2
were first treated to a solution containing 50 μM ligand (AQP4 and
AQDA1−3). The spectra were recorded after 10 min incubation at
room temperature. The Fe(II) samples were prepared in an anaerobic
N -filled glovebox. CuCl (50 μM) was then added to a solution of
2
2
compound and a divalent metal chloride salt. The spectra were taken
after an additional 10 min incubation period at room temperature.
Quantification of metal selectivity was calculated by comparing and
normalizing the absorption values of metal−ligand complexes at 290
0
0
supernatant of cell lysates) and was plotted as a function of compound
concentration. The TEAC value of ligands was calculated as a ratio of
the slope of the standard curve of the compound to that of Trolox.
The measurements were conducted in triplicate.
(
for AQP4), 440 (for AQDA1 and AQDA2), and 338 nm (for
AQDA3) to the absorption at these wavelengths before and after the
addition of CuCl (A /A ).
2
M
Cu
Solution Speciation Studies. The pK values for AQP1, AQP4,
Parallel Artificial Membrane Permeability Adapted for the
Blood−Brain Barrier (PAMPA-BBB) Assay. PAMPA-BBB experi-
ments were carried out using the PAMPA Explorer kit (pION Inc.,
Billerica, MA) with modifications to previously reported proto-
a
and AQDA1−3 were determined through UV−vis variable-pH
2
3,31,32,35
titrations based on a previously reported procedure.
To
obtain pK values for the ligands (50 μM for AQP4 or AQDA1; 25
a
2
3,25,26,55
μM for AQP1, AQDA2, or AQDA3), HCl was titrated into the
speciation solution (100 mM NaCl, pH 12, 10 mM NaOH) in small
aliquots to obtain at least 30 spectra in the range pH 2−11 (for AQP1
and AQP4) or pH 2−10 (for AQDA1−3). In addition, to investigate
Cu(II) binding to the ligands at various pHs, small aliquots of HCl
were titrated into the solutions containing a ligand and a metal
cols.
Each stock solution was diluted with Prisma HT buffer
(pH 7.4, pION) to a final concentration of 25 μM (1% v/v final
DMSO concentration). The resulting solution was added to wells of
the donor plate (200 μL, 12 replicates). BBB-1 lipid formulation (5
μL, pION) was used to coat the polyvinylidene fluoride (PVDF, 0.45
mM) filter membrane on the acceptor plate. This acceptor plate was
placed on top of the donor plate forming a sandwich. Brain sink buffer
(BSB, 200 μL, pION) was added to each well of the acceptor plate.
The sandwich was incubated for 4 h at ambient temperature without
stirring. UV−vis spectra of the solutions in the reference, acceptor, and
donor plates were measured using a microplate reader. The PAMPA
chloride salt [[M(II)]:[L] = 1:2; [CuCl ] = 50 (for AQP4), 12.5 (for
2
AQDA1 and AQDA2), and 25 μM (for AQP1 and AQDA3)]. At least
3
0 spectra were measured over the range pH 2−8. The acidity and
stability constants were calculated by using the HypSpec program
52,53
(
Protonic Software, Leeds, U.K.).
-Deoxyribose Assay. The ability of AQ1, AQ3, AQ4, AQDA1,
2
Explorer software v. 3.5 (pION) was used to calculate −logP for each
e
and AQDA3 to suppress the generation of hydroxyl radicals was
compound. CNS± designations were assigned by comparison to
2
5,26,55
determined by the 2-deoxyribose assay. The assay was preformed on
compounds that were identified in previous reports.
23,36
the basis of previously reported methods.
Chelexed solutions were
Cell Viability Measurements. The mouse Neuro-2a (N2a)
neuroblastoma cell line was purchased from the American Type Cell
Collection (ATCC, Manassas, VA). Cells were maintained in media
containing 1:1 DMEM (GIBCO, Grand Island, NY) and opti-MEM
(GIBCO), supplemented with 5% (v/v) fetal bovine serum (FBS;
GIBCO), 1% (v/v) L-glutamine (GIBCO), 100 U/mL penicillin, and
100 mg/mL streptomycin (GIBCO). The cells were grown and
used, and reactions (total volume, 200 μL) were prepared by mixing,
in the following order, buffer (50 mM NaH PO , pH 7.4), ligand (125
2
4
μM), CuCl (10 μM), 2-deoxy-D-ribose (15 mM), H O (200 μM),
2
2
2
and sodium ascorbate (2 mM) and allowed to react for 1 h at 37 °C
with constant agitation. The reactions were quenched upon addition of
trichloroacetic acid (200 μL of 2.8% m/v) and 2-thiobarbituric acid
L
DOI: 10.1021/acs.inorgchem.6b00525
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
maintained at 37 °C in a humidified atmosphere with 5% CO . Cell
(13) Corbett, A.; Pickett, J.; Burns, A.; Corcoran, J.; Dunnett, S. B.;
Edison, P.; Hagan, J. J.; Holmes, C.; Jones, E.; Katona, C.; Kearns, I.;
Kehoe, P.; Mudher, A.; Passmore, A.; Shepherd, N.; Walsh, F.; Ballard,
C. Nat. Rev. Drug Discovery 2012, 11, 833−846.
2
viability upon treatment with compounds was determined by the MTT
assay [MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-
lium bromide, Sigma-Aldrich]. N2a cells were seeded in a 96 well plate
(
15 000 cells in 100 μL per well). The cells were treated with
(14) Rodríguez-Rodríguez, C.; Telpoukhovskaia, M.; Orvig, C. Coord.
Chem. Rev. 2012, 256, 2308−2332.
compounds (5 or 10 μM, 1% v/v final DMSO concentration) with or
without CuCl or ZnCl (5 or 10 μM), and incubated for 24 h. After
(15) Schugar, H.; Green, D. E.; Bowen, M. L.; Scott, L. E.; Storr, T.;
2
2
incubation, MTT [25 μL; 5 mg/mL in phosphate buffered saline
PBS, pH 7.4, GIBCO)] was added to each well, and the plate was
Bohmerle, K.; Thomas, F.; Allen, D. D.; Lockman, P. R.; Merkel, M.;
̈
(
Thompson, K. H.; Orvig, C. Angew. Chem., Int. Ed. 2007, 46, 1716−
incubated for 4 h at 37 °C. Formazan produced by the cells was
solubilized using an acidic solution of N,N-dimethylformamide (DMF,
1
718.
(
16) Lincoln, K. M.; Offutt, M. E.; Hayden, T. D.; Saunders, R. E.;
Green, K. N. Inorg. Chem. 2014, 53, 1406−1416.
17) Lincoln, K. M.; Gonzalez, P.; Richardson, T. E.; Julovich, D. A.;
Saunders, R.; Simpkins, J. W.; Green, K. N. Chem. Commun. 2013, 49,
712−2714.
18) Lincoln, K. M.; Richardson, T. E.; Rutter, L.; Gonzalez, P.;
Simpkins, J. W.; Green, K. N. ACS Chem. Neurosci. 2012, 3, 919−927.
19) Otto, R.; Penzis, R.; Gaube, F.; Adolph, O.; Fohr, K. J.;
5
0%, v/v aq) and sodium dodecyl sulfate (SDS, 20%, w/v) overnight
at room temperature in the dark. The absorbance was measured at 600
nm using a microplate reader. Cell viability was calculated relative to
cells treated with an equivalent amount of DMSO. All experiments
were performed in triplicate.
(
2
(
ASSOCIATED CONTENT
Supporting Information
(
̈
■
*
S
Warncke, P.; Robaa, D.; Appenroth, D.; Fleck, C.; Enzensperger, C.;
Lehmann, J.; Winckler, T. J. Med. Chem. 2015, 58, 6710−6715.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b00525.
(20) Telpoukhovskaia, M. A.; Cawthray, J. F.; Rodríguez-Rodríguez,
C.; Scott, L. E.; Page, B. D. G.; Patrick, B. O.; Orvig, C. Bioorg. Med.
Chem. Lett. 2015, 25, 3654−3657.
Syntheses, docking investigations, cytotoxicity investiga-
tions, additional gel/Western blot, TEM, NMR, mass
spectrometry, and metal binding experiments (PDF)
(21) Storr, T.; Merkel, M.; Song-Zhao, G. X.; Scott, L. E.; Green, D.
E.; Bowen, M. L.; Thompson, K. H.; Patrick, B. O.; Schugar, H. J.;
Orvig, C. J. Am. Chem. Soc. 2007, 129, 7453−7463.
(22) Wang, Z.; Wang, Y.; Li, W.; Mao, F.; Sun, Y.; Huang, L.; Li, X.
ACS Chem. Neurosci. 2014, 5, 952−962.
AUTHOR INFORMATION
Corresponding Author
E-mail: mhlim@unist.ac.kr.
■
(
23) Lee, S.; Zheng, X.; Krishnamoorthy, J.; Savelieff, M. G.; Park, H.
M.; Brender, J. R.; Kim, J. H.; Derrick, J. S.; Kochi, A.; Lee, H. J.; Kim,
C.; Ramamoorthy, A.; Bowers, M. T.; Lim, M. H. J. Am. Chem. Soc.
*
2
(
014, 136, 299−310.
24) Savelieff, M. G.; DeToma, A. S.; Derrick, J. S.; Lim, M. H. Acc.
Chem. Res. 2014, 47, 2475−2482.
25) Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T.
Eur. J. Med. Chem. 2003, 38, 223−232.
26) Avdeef, A.; Bendels, S.; Di, L.; Faller, B.; Kansy, M.; Sugano, K.;
Notes
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
■
This work was supported by the University of Michigan Protein
Folding Disease Initiative (to A.R., B.T.R., and M.H.L.) and the
National Research Foundation of Korea (NRF) grant funded
by the Korean government [NRF-2014S1A2A2028270 (to
M.H.L. and A.R.); NRF-2014R1A2A2A01004877 (to
M.H.L.)], and the 2016 Research Fund (Project Number
(
Yamauchi, Y. J. Pharm. Sci. 2007, 96, 2893−2909.
(27) Lim, M. H.; Wong, B. A.; Pitcock, W. H.; Mokshagundam, D.;
Baik, M.-H.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 14364−14373.
(
28) Waibel, M.; Hasserodt, J. Tetrahedron Lett. 2009, 50, 2767−
2
769.
29) Roth, R.; Erlenmeyer, H. Helv. Chim. Acta 1954, 37, 1064−
068.
30) Shaffer, K. J.; Davidson, R. J.; Burrell, A. K.; McCleskey, T. M.;
Plieger, P. G. Inorg. Chem. 2013, 52, 3969−3975.
31) Derrick, J. S.; Kerr, R. A.; Nam, Y.; Oh, S. B.; Lee, H. J.; Earnest,
(
1
(
1
.160001.01) of Ulsan National Institute of Science and
Technology (UNIST). J.K. acknowledges the support from
the Global Ph.D. fellowship program through the National
Research Foundation of Korea (NRF) funded by the Ministry
of Education (NRF-2015HIA2A1030823).
(
K. G.; Suh, N.; Peck, K. L.; Ozbil, M.; Korshavn, K. J.; Ramamoorthy,
A.; Prabhakar, R.; Merino, E. J.; Shearer, J.; Lee, J.-Y.; Ruotolo, B. T.;
Lim, M. H. J. Am. Chem. Soc. 2015, 137, 14785−14797.
(32) Beck, M. W.; Oh, S. B.; Kerr, R. A.; Lee, H. J.; Kim, S. H.; Kim,
S.; Jang, M.; Ruotolo, B. T.; Lee, J.-Y.; Lim, M. H. Chem. Sci. 2015, 6,
1879−1886.
(33) Hernandez, H.; Robinson, C. V. Nat. Protoc. 2007, 2, 715−726.
́
(34) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S.-
J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139−1152.
(35) Hyung, S.-J.; DeToma, A. S.; Brender, J. R.; Lee, S.;
Vivekanandan, S.; Kochi, A.; Choi, J.-S.; Ramamoorthy, A.; Ruotolo,
B. T.; Lim, M. H. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3743−3748.
(36) Charkoudian, L. K.; Pham, D. M.; Franz, K. J. J. Am. Chem. Soc.
2006, 128, 12424−12425.
(37) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.;
Rice-Evans, C. Free Radical Biol. Med. 1999, 26, 1231−1237.
(38) Puskullu, M. O.; Tekiner, B.; Su
REFERENCES
■
(
1) Guzior, N.; Wieckowska, A.; Panek, D.; Malawska, B. Curr. Med.
Chem. 2015, 22, 373−404.
2) Perez, L. R.; Franz, K. J. Dalton Trans. 2010, 39, 2177−2187.
3) Zatta, P.; Drago, D.; Bolognin, S.; Sensi, S. L. Trends Pharmacol.
(
(
Sci. 2009, 30, 346−355.
(4) Duce, J. A.; Bush, A. I. Prog. Neurobiol. 2010, 92, 1−18.
(5) Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8, 101−112.
(6) Jakob-Roetne, R.; Jacobsen, H. Angew. Chem., Int. Ed. 2009, 48,
3
(
(
030−3059.
7) Scott, L. E.; Orvig, C. Chem. Rev. 2009, 109, 4885−4910.
8) Miller, Y.; Ma, B.; Nussinov, R. Chem. Rev. 2010, 110, 4820−
4838.
(
9) Telpoukhovskaia, M. A.; Orvig, C. Chem. Soc. Rev. 2013, 42,
836−1846.
10) Jensen, M.; Canning, A.; Chiha, S.; Bouquerel, P.; Pedersen, J.
T.; Østergaard, J.; Cuvillier, O.; Sasaki, I.; Hureau, C.; Faller, P. Chem.
1
(
̈
zen, S. Mini-Rev. Med. Chem.
2013, 13, 365−372.
(39) Havsteen, B. H. Pharmacol. Ther. 2002, 96, 67−202.
(40) Rice-Evans, C.; Miller, N. J.; Paganga, G. Free Radical Biol. Med.
1996, 20, 933−956.
-
Eur. J. 2012, 18, 4836−4839.
(
11) Kepp, K. P. Chem. Rev. 2012, 112, 5193−5239.
(
12) Derrick, J. S.; Lim, M. H. ChemBioChem 2015, 16, 887−898.
(41) Mruk, D. D.; Cheng, C. Y. Spermatogenesis 2011, 1, 121−122.
M
DOI: 10.1021/acs.inorgchem.6b00525
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(
42) Schanda, P.; Brutscher, B. J. Am. Chem. Soc. 2005, 127, 8014−
015.
43) Vivekanandan, S.; Brender, J. R.; Lee, S. Y.; Ramamoorthy, A.
Biochem. Biophys. Res. Commun. 2011, 411, 312−316.
44) Yoo, S. I.; Yang, M.; Brender, J. R.; Vivekanandan, S.; Sun, K.;
8
(
(
Joo, N. E.; Jeong, S.-H.; Ramamoorthy, A.; Kotov, N. A. Angew. Chem.,
Int. Ed. 2011, 50, 5110−5115.
(
45) Fawzi, N. L.; Ying, J.; Torchia, D. A.; Clore, G. M. J. Am. Chem.
Soc. 2010, 132, 9948−9951.
(
(
(
46) Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455−461.
47) Wolf, C. K. Chem. Eng. News 2009, 87, 31.
48) Giles, K.; Williams, J. P.; Campuzano, I. Rapid Commun. Mass
Spectrom. 2011, 25, 1559−1566.
49) Zhong, Y.; Hyung, S.-J.; Ruotolo, B. T. Analyst 2011, 136,
534−3541.
50) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.;
Ruotolo, B. T. Anal. Chem. 2010, 82, 9557−9565.
51) Soper, M. T.; DeToma, A. S.; Hyung, S.-J.; Lim, M. H.; Ruotolo,
(
3
(
(
B. T. Phys. Chem. Chem. Phys. 2013, 15, 8952−8961.
(
(
52) Gans, P.; Sabatini, A.; Vacca, A. Ann. Chim. 1999, 89, 45−49.
53) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca,
A. Coord. Chem. Rev. 1999, 184, 311−318.
54) Spencer, V. A.; Sun, J.-M.; Li, L.; Davie, J. R. Methods 2003, 31,
7−75.
55) BBB Protocol and Test Compounds; pION Inc.: Woburn, MA,
009.
(
6
(
2
N
DOI: 10.1021/acs.inorgchem.6b00525
Inorg. Chem. XXXX, XXX, XXX−XXX