Enhancement of the Antioxidant Activity and Neurotherapeutic Features through Pyridol Addition to Tetraazamacrocyclic Molecules

Article abstract of DOI:10.1021/acs.inorgchem.9b02932

Alzheimer's and other neurodegenerative diseases are chronic conditions affecting millions of individuals worldwide. Oxidative stress is a consistent component described in the development of many neurodegenerative diseases. Therefore, innovative strategi

Full text of DOI:10.1021/acs.inorgchem.9b02932

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Enhancement of the Antioxidant Activity and Neurotherapeutic
Features through Pyridol Addition to Tetraazamacrocyclic
Molecules
Hannah M. Johnston,† Kristof Pota,† Madalyn M. Barnett,† Olivia Kinsinger,‡ Paige Braden,‡
Timothy M. Schwartz,† Emily Hoffer,† Nishanth Sadagopan,† Nam Nguyen,† Yu Yu,§ Paulina Gonzalez,†
∥
Gyula Tircso, Hongli Wu,§,⊥ Giridhar Akkaraju,‡ Michael J. Chumley,‡ and Kayla N. Green*,†
́
†Department of Chemistry and Biochemistry and ‡Department of Biology, Texas Christian University (TCU), 2950 S. Bowie, Fort
Worth, Texas 76129, United States
⊥
§Pharmaceutical Sciences, University of North Texas System College of Pharmacy, and North Texas Eye Research Institute,
University of North Texas (UNT) Health Science Center, Fort Worth, Texas 76107, United States
∥
́
Department of Inorganic and Analytical Chemistry, Faculty of Science and Technology, University of Debrecen, Egyetem ter 1,
Debrecen H-4010, Hungary
S
* Supporting Information
ABSTRACT: Alzheimer’s and other neurodegenerative dis-
eases are chronic conditions affecting millions of individuals
worldwide. Oxidative stress is a consistent component
described in the development of many neurodegenerative
diseases. Therefore, innovative strategies to develop drug
candidates that overcome oxidative stress in the brain are
needed. To target these challenges, a new, water-soluble 12-
membered tetraaza macrocyclic pyridinophane L4 was
designed and produced using a building-block approach.
Potentiometric data show that the neutral species of L4
provides interesting zwitterionic behavior at physiological pH,
akin to amino acids, and a nearly ideal isoelectric point of 7.3.
The copper(II) complex of L4 was evaluated by X-ray diffraction and cyclic voltammetry to show the potential modes of
antioxidant activity derived, which was also demonstrated by 2,2-diphenyl-1-picrylhydrazyl and coumarin carboxylic acid
antioxidant assays. L4 was shown to have dramatically enhanced antioxidant activity and increased biological compatibility
compared to parent molecules reported previously. L4 attenuated hydrogen peroxide (H2O2)-induced cell viability loss more
efficiently than precursor molecules in the mouse hippocampal HT-22 cell model. L4 also showed potent (fM) level protection
against H2O2 cell death in a BV2 microglial cell culture. Western blot studies indicated that L4 enhanced the cellular antioxidant
defense capacity via Nrf2 signaling activation as well. Moreover, a low-cost analysis and high metabolic stability in phase I and II
models were observed. These encouraging results show how the rational design of lead compounds is a suitable strategy for the
development of treatments for neurodegenerative diseases where oxidative stress plays a substantial role.
when there is an imbalance between the reactive oxygen
species (ROS) and the availability/activity of antioxidants.5
The brain requires disproportionally large amounts of
molecular oxygen for processes including cellular respiration
and biosynthesis of neurotransmitters, compared to other
organs of similar size. Redox-active transition-metal ions are
also vital components of the brain that are essential for
detoxification of free radicals, electron- and oxygen-transport
processes, neurotransmitter biosynthesis, and neuronal signal-
ing. These processes are elegantly facilitated through metal
uptake and transport mechanisms as well as metal coordination
INTRODUCTION
■
Alzheimer’s and other neurodegenerative diseases are chronic
conditions affecting millions of individuals worldwide.1,2
Despite aggressive efforts, there are yet to be therapeutic
agents approved for and capable of eradicating these diseases.3
The deficiency of clearly understood mechanisms for the cause
and progression of neurodegenerative diseases is a major
impediment to developing a treatment. However, a few main
themes consistently arise and are proposed to be interrelated in
many of these diseases: increased oxidative stress,4−9
dyshomeostasis of metal ions,10−12 and protein aggrega-
tion.13−17
The human brain is particularly susceptible to protein, lipid,
Received: October 2, 2019
and DNA damage derived from the oxidative stress that occurs
© XXXX American Chemical Society
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in protein scaffolds or cofactors.18 However, disruption of
these systems is linked to the increased production of ROS.
Unregulated redox-active metals, like copper and iron, can
readily react with molecular oxygen and biological reductants
such as ascorbate to produce excess ROS via Fenton- and
Haber−Weiss-type reactions.19 Aberrant metal ions promote
β-amyloid (Aβ) aggregation, a causative agent in Alzheimer’s
disease. Disproportionately high levels of metal ions have been
observed in the hallmark Aβ plaques, are known to promote
peptide aggregation, and provide an environment that also
promotes the redox-active environment responsible for ROS
production.10,12,14,20−22 The relationship between disease
causation and the progression of metal-ion misregulation,
oxidative stress, and protein deposition is a challenge to
untangle.10,23 This work aims to focus on the development of a
new, potent small molecule that targets two legs of the “piano
stool” (Figure 1a) that contribute to neurodegenerative
disease: oxidative stress and metal-ion misregulation.
number of donor atoms, would result in an increase of the
radical scavenging ability and antioxidant activity, as well as
improve on the isoelectric point compared to other antioxidant
macrocycles reported to date.52 The design of the new
molecule (L4) would preserve the water solubility, metabolic
stability, and redox control of metal ions. While the Py2N2
family of molecules based on the 2,11-diaza[3,3](2,6)-
pyridinophane ligand have been explored extensively for
catalytic reactions, the bispyridol L4 congener is reported
here for the first time.25,27,29,32,33,41 Potentiometric titrations
were performed with L4 to determine the pKa and pI values,
which were compared to precursor molecules. Coumarin
carboxylic acid (CCA) and 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH•) assays were used to preliminarily evaluate
the antioxidant capabilities of L4. In addition to these assays,
L4 has been coordinated to copper(II), which has been
associated with the development of Alzheimer’s disease.53−60
The CuL4 complex was characterized using X-ray diffraction
(XRD) and cyclic voltammetry (CV). HT-22 cellular studies
indicate that L4 is a superior antioxidant to L2, and the NRF2
pathway was upregulated to give rise to cell protection. Potent
protection against hydrogen peroxide (H2O2)-induced cell
death was also observed in a BV microglial cell (MGC) culture.
These measurements, metabolism (phases I and II), cell
toxicity, and other antioxidant studies encourage further
exploration of L4 as well as other derivations of pyridino-
phanes as antioxidant therapeutic agents for neurodegenerative
diseases.
RESULTS AND DISCUSSION
■
Synthesis of L4. The synthesis of L4 was achieved via a 1:1
condensation reaction (Scheme 1). A total of 2 equiv of
tosylamide monosodium salt (TsNHNa)61−64 was combined
with 4-benzyloxy-2,6-bis(chloromethyl)pyridine (1)50,65 in
N,N-dimethylformamide (DMF) under reflux to produce the
cyclized and tosyl-protected product 8,17-benzyloxy-4,13-
bis(p-toluenesulfonyl)-1,4,11,13-tetraazabis(2,6-pyridino-
phane) (2). This cyclization reaction can be visualized as
proceeding through a monoalkylated intermediate followed by
self-condensation in the presence of excess TsNHNa, which
acts as a base, to yield the desired macrocyclic product
(Scheme S1).63 A low-quality solid-state structure of 2
obtained through XRD analysis (Figures S1 and S2) is
observed in the lowest-energy syn chair−chair conformation.
Concomitant removal of the benzyl and tosyl protecting
groups was accomplished with a naphthalene-catalyzed
lithiation (Scheme 1).66,67 The reaction mixture was then
hydrolyzed and an acidic workup yielded L4 as the
trihydrochloride salt. NMR analysis was consistent with the
Figure 1. (a) Causative agents for neurodegenerative diseases and (b)
structures of L1−L4 and reactivities observed to date.
Pyridinophane molecules based on L1 (Figure 1b) have
been used as metal-binding scaffolds that support a range of
catalytic reactions and serve as the backbone for potential
magnetic resonance imaging contrast agents.24−49 We have
also reported applications of 12-membered pyridinophane
molecules L1−L3 (Figure 1b) as potential therapeutics for
neurodegenerative diseases derived from oxidative stress.50,51
Such work inspired the synthesis and exploration of the new
water-soluble, more-active, and biologically compatible system
L4 described herein.
With the goal of increasing the potency and thus lowering
the effective dosage, it was hypothesized that doubling the
pyridol moieties of L2, while retaining the macrocycle size and
Scheme 1. Synthetic Methods Used To Produce L4
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connectivity shown in Scheme 1. The acid form of the ligand is
highly soluble in water.
Protonation Constants of L4. Pharmacological behaviors,
solubility, permeability, log D, and oral absorption can be
predicted or explained by taking into account the protonation
state of a complex at biological pH. These factors are strongly
modulated by the distribution of neutral and charged species
for a selected compound. Therefore, the stepwise protonation
constants (log KiH) of L4 were determined by pH-
potentiometric titrations at constant ionic strength and
temperature (I = 0.15 M NaCl and T = 298 K). The results
are compiled in Table 1, along with the protonation constants
a
Table 1. Protonation Constants (pKa) and pI Values of
Studied Cyclen and Pyridinophanes L1, L2, and L4 (I = 0.15
M NaCl and T = 298 K)
b
c
c
cyclen
L1
L2
L4
H
H
H
H
H
log K1
log K2
log K3
log K4
log K5
10.66
9.69
1.41
11.37
8.22
1.61
11.56
9.05
5.45
1.68
11.307(8)
9.35(2)
5.25(3)
4.21(3)
0.98(2)
31.10
Figure 2. Proposed protonation sequence and equilibrium distribu-
tion diagram with absorbance values at 258 nm from UV
spectrophotometric titration for L4 (cL = 0.02 mM, I = 0.15 M
NaCl, and T = 298 K).
∑log KiH
pI
21.76
>10.66
21.20
>11.37
27.74
10.31
species detectable in the experimental window. The presence
of two aromatic downfield resonances suggested an asym-
metric form of L4 with negative charges on one of the pyridol
O atoms and the N atom of the other pyridol moiety (Figure 2,
top). The most stable resonance structure of [L4]2− would
contain both enol and keto functionalities opposite to one
another. The second protonation step (pKa = 9.35) is assigned
to a secondary amine, based on the observed upfield shifts to
the aliphatic derived H atoms and no change in the downfield
resonances. The third protonation event gives rise to
significant changes in both the most downfield aliphatic (red
squares) and aromatic (dark-blue circles) proton resonances of
L4 (Figure 3), consistent with protonation of the enol group.
7.30
a
Defined as KiH = [HiLi+]/([H+][Hi−1L(i−1)+]) for i = 1−5.
Reference 68 (I = 0.15 M NaClO4). Reference 52.
b
c
previously reported for cyclen and L1 and L2.52,69−71
A
thorough comparison of the impact of the position of −OH on
pKa (L2 vs L3) is reported elsewhere.52 For each macrocycle
compared here, all N atoms are expected to undergo
protonation equilibria but may not be measurable in the
window of the potentiometric experiment. Furthermore,
ligands L2 and L4 also contain hydroxyl groups on the 4
position of the pyridine ring also capable of protonation
equilibria.
Five protonation constants were measured for L4 (Table 1)
within the window of the experiment. With the addition of
each pyridol moiety (L1 < L2 < L4), the ligands exhibit an
increase in the values measured for the second protonation
step.52 This indicates that the presence of an −OH group on
the pyridine ring increases the basicity of the ligand, which is
consistent with the enhanced resonance stabilization imparted
by the electron-donating −OH moiety on the 4 position of the
pyridine ring.52
The results indicate that at pH 7.3 the [H2(L4)] species will
dominate at 98%. In fact, this neutral species is modeled to be
>50% between pH values of ∼6 and 9, an unusually broad
range (Figure 2). Moreover, the isoelectric point of L4 (pI =
7.3) is ideal for drug candidates.72−74 This is an improvement
over L2 with pI = 10.31 and shows another advantage of using
the additional pyridol moiety of L4 to tune pharmacological
features of a potential drug.75 The pI value is consistent with
the water solubility observed and indicates that L4 could
exhibit positive pharmaceutical properties. To gain more
insight into the most stable resonance structure of L4 at
1
Figure 3. H NMR titration curves of L4.
The small decrease in the UV−vis spectrum indicates that no
large changes in the electronic structure occurred at this
protonation step. However, the next protonation (pKa = 4.21)
yields a significant decrease in the aromatic band of L4 (258
nm) concomitant with similar downfield shifts in the adjacent
aromatic (green diamonds) and aliphatic (purple triangles)
resonances compared to those observed with the previous
protonation event. These observations are consistent with
transitioning from an 8-electron/7-centered system (keto) to
an aromatic 6-electron/6-centered system (enol). Only one
1
physiological pH, H NMR spectroscopy and UV spectropho-
tometric titrations were carried out. Elemental analysis
indicated that L4 was isolated as the trihydrochloride salt
salt, which provided a starting point for assigning the
protonation sequence. Five pKa values were observed.
Therefore, the dianionic form was the most deprotonated
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Figure 4. Radical scavenging ability measured using DPPH• versus L2 and L4 compared to the standard antioxidant BHT (n = 3). The results have
been standardized against DPPH• with no radical scavenger present (100%).
aliphatic resonance is observed at pH values below pD = 3.
This can be explained by rearrangement of the proton (red,
Figure 3, top) from the pyridine N atom to the remaining
unprotonated secondary amine. The last protonation step
modeled from the potentiometric data was not observable
under the conditions used in the UV or NMR studies.
However, further acidification of a sample of L4 resulted in
neurodegenerative disorders.82−84 Selective reactivity with
either ABTS or DPPH• has been observed for other
antioxidant molecules but remains a topic of debate regarding
mechanistic pathways.85−90 Nevertheless, the percentage of
DPPH• quenched by L4 is consistently >50% more than that
of L2 and strongly supports the hypothesis that doubling the
number of pyridol groups from L2 would drastically increase
the radical scavenging activity.
1
only two resonances in the H NMR spectrum (Figure S3).
Prevention of ROS Generation via Redox Cycling.
ROS can be produced by the redox cycling of redox-active
transition-metal ions.79 The overproduction of ROS is
hypothesized to lead to the development of neurodegenerative
diseases.91−94 A good model for such redox cycling in the brain
is the copper/ascorbate redox system (Scheme 2).50,95,96 The
The symmetric bisenol form of L4 is consistent with the last
acidic equivalent residing equally between the two pyridine N
atoms of [H5(L4)]3+, which agrees with previously reported
assignments of pyridinophanes.52,76 These results highlight the
need to carefully study the protonation events and indicate that
the [H2L4] species would be observed as a highly asymmetric
species at physiological pH, which parallels the zwitterionic
behavior of amino acids. This is in contrast to the symmetric
trihydrochloride version in which it is isolated.
Scheme 2. Copper(II)/Copper(I) Redox Cycling Occurring
in the Presence of Oxygen and Ascorbate To Produce ROS
Radical Scavenging Ability. The strong correlation
between uncontrolled oxidative stress and the development
of neurodegenerative diseases has led to the study of a range of
antioxidant small molecules and was the premise for the
exploration of L4.77 DPPH• (λmax = 520 nm) was used to assay
the radical scavenging activity of L4 compared to L2.78−81
A
radical scavenging molecule can quench DPPH•, and the
corresponding color change from purple to clear yellow can be
monitored spectrophotometrically to evaluate the radical
reactivity (Scheme S2).78−81 The concentrations of L2 (one
pyridol moiety) and L4 (two pyridol moieties) were studied
over the range of 1−1500 μM (Figure 4). 2,6-Di-tert-butyl-4-
methylphenol, or butylated hydroxytoluene (BHT), was used
as a positive control and resulted in a rapid decrease in the
DPPH• absorbance band.78,79 Likewise, L4 showed a strong,
concentration-dependent antioxidant response beginning at
100 μM (Figure 4). The lack of radical scavenging activity
exhibited by L1 was previously shown and confirms that the
pyridol moiety is responsible for the strong response exhibited
with L2 and L4.50 The reactivities of L2 and L4 were also
tested with 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic
acid (ABTS), but little to no reactivity was observed. This
suggests that the radical scavenging activity is divergent from
molecules reported by others for the potential treatment of
hydroxyl radicals generated by copper(II) in the presence of
ascorbate and oxygen stoichiometrically convert CCA to the
fluorescent hydroxy-CCA species (Figure 5, inset).95,96 Metal-
ion redox chemistry is known to be modulated by coordination
to strongly donating small molecules.19,97 Therefore, this
model system was used to evaluate the capacity of L4 to halt
metal-ion redox cycling under these conditions. The results are
compared to L2 to determine if the number of pyridol moieties
may play a role in this reactivity as well.
The addition of 1 equiv of L4 [vs copper(II) concentration]
resulted in an almost complete inhibition of CCA conversion
to fluorescent hydroxy-CCA. To show the stoichiometric
nature of this response, 0.5 equiv of each metal-binding
molecule was added in a follow-up study. The fluorescent
signal observed was greater than the samples incubated with 1
equiv but less than 50% of that compared to the sample sans a
metal-binding molecule.
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Figure 5. Fluorescence intensity of 7-hydroxy-CCA after incubation of CCA (100 μM) and ascorbate (300 μM) with copper(II) (10 μM).
Compounds L2 (0.5 equiv = 5 μM; 1 equiv = 10 μM) and L4 (0.5 equiv = 5 μM; 1 equiv = 10 μM) were combined prior to the addition of
ascorbate. No fluorescence was observed with CCA coincubated with samples of (1) ascorbate (300 μM) only or (2) copper(II) (10 μM). All
solutions except CuSO4·6H2O (Milli-Q water only) were dissolved and diluted in a pH = 7.4 1X KH2PO4/NaCl buffer containing Desferal (1 μM).
Final volume = 3 mL (n = 3). The inset shows the response of CCA with no metal-binding agent added.
This prevention of OH• formation was hypothesized to be a
result of the molecule’s ability to accommodate copper(II) but
not copper(I) because of its rigidity. To test this, the open-
chain, NNN-pincer 2,6-bis[(diethylamino)methyl]pyridine
was produced using methods described by Vedernikov et al.
and subsequently evaluated with CCA assay.98 One full
equivalent of the open-chain system, with respect to the
copper(II) concentration, failed to give any indication of the
prevention of copper redox cycling and the formation of OH•
(Figure S6). This is consistent with pincer-type ligands that
readily accommodate CuI ions. Furthermore, electrochemical
analysis of the CuL4 complex shows one irreversible reduction
at very negative potential values (Epc = −816 mV vs Fc+/Fc in
DMF; Figure S7). The position of the copper(II) to copper(I)
reduction is significantly more negative that of the copper(II/
I)/ascorbate reaction, thus indicating that the macrocycle
stabilized the copper(II) species. Therefore, the activity
observed with macrocycles to date, including L4, is a result
of the rigidity of the macrocycle preventing the preferred
geometric environment for copper(I). The lack of fluorescence
observed within the CCA/copper/ascorbate system is hence
attributed to the chelation of CuII ions by L4.
Interestingly, L4 is slightly more efficient compared to L2
for the prevention of hydroxyl-CCA formation. The enhanced
ability of L4 to halt redox cycling is attributed to the additional
pyridol moiety, which may quench any hydroxyl radicals
produced by copper(II) before interaction with the metal-
binding molecules. Most importantly, both L2 and L4 exhibit
the ability to halt the redox cycling of copper(II) and ascorbate
in an aerobic environment, making molecules of this type good
candidates for use as antioxidant therapeutics.
Copper(II)-Binding Studies. The results of L4 with CCA
assay and CV suggest that L4 is an effective metal-binding
small molecule. Py2N2 systems have been studied extensively
with metals such as manganese(II) and manganese(III) but
have not been explored with copper(II) or compared with
congeners like L1 and L2.32,33,41,49 Therefore, the copper
complex of L4 was produced and evaluated by XRD analysis. A
total of 1 equiv of copper(II) perchlorate was added dropwise
to a solution of L4 at pH ∼ 5 (Figure 6a). The aqueous
Figure 6. (a) Complexation reaction of L4 with copper(II) and the
resulting molecular structure of CuL4 with an atom-labeling scheme
and counterions removed for clarity. (b) Molecular structure of the
monomeric unit of CuL4 highlighting the interactions between the
aromatic groups.
solution of L4 changed from pale yellow to light blue upon the
addition of copper(II). Light-green crystalline material suitable
for XRD analysis was obtained by several cycles of slow
evaporation of methanol (MeOH) solutions. The resulting
molecular structure (Figure 6b) modeled in the P21/c space
group shows a molecular formula of [CuL4Cl]2[Cl]2 for the
copper(II) complex of L4 in the solid state. Each L4 molecule
binds to a CuII ion in a syn boat−boat fashion (Figure S1),
with the two pyridine N atoms in the equatorial plane and the
remaining two secondary N atoms in the axial plane. Hence,
L4 generates a 5−5−5−5 ring structure when bound to the
CuII center. The coordination environment of each CuL4
monomeric unit is completed by two μ-Cl ions bridging
between the CuII centers.
The cis-α coordination generated by the 12-membered
pyridinophane macrocycle is consistent with the precursors in
the literature.99 However, the dimeric structure is unique for
this copper complex. We hypothesize that the dimeric
structure is a result of the highly symmetric nature of the
complex in the solid state and have no evidence that it persists
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in solution. The monomeric form is expected to be produced
upon dissolution, particularly in coordinating solvents such as
water. This assignment is supported by the monomeric species
[CuL4Cl]+ observed in the mass spectrometry (MS) spectrum
of CuL4 (Figure S8). The cis-α coordination orients the
pyridol rings within L4 to potentially interact with one
another. In fact, the distance between the pyridol ring
centroids is 4.117 Å (Figure 6), and the angle between the
rings (62.36°) is within the parameters for an edge-to-face π−π
interaction, which are defined as interactions between two
aromatic rings in which the angle between the ring planes is
between 60° and 120° and the distance between the ring
centroids is less than 5.5 Å. Altogether, the coordination
chemistry of CuL4 is consistent with the results of CCA assay,
which indicates a stable copper(II)−L4 interaction with a
typical bonding arrangement for the pyridinophane-type
molecules.
Therapeutic Window of L4. Cell toxicity studies were
carried out to evaluate the potential for L4 to serve as a
therapeutic agent and to establish a therapeutic window for
future in vivo studies. HT-22 hippocampal neuronal cells were
used as a model, and an IC50 value of 566.4 μM was observed
for L4. The toxicity of L4 was compared to those of L1, L2,
and cyclen (Figure 7). The order of toxicity within the series is
order of toxicity parallels that of the pI (Table 1) of each
complex, suggesting that this feature may play a role as well.
However, the correlation between pI and toxicity would need
to be further explored in future work to validate this.
Antioxidant Protection in Neuronal Cells and MGCs.
To investigate the cytoprotective effects of L4, HT-22 cells
(neuronal) were pretreated with or without L4 (0.1−100 μM)
for 24 h, followed by the addition of 600 μM H2O2 for 4 h. As
shown in Figure 8, H2O2 treatment alone caused approx-
imately 50% cell viability loss in HT-22 cells. For comparison,
pretreatment with 1 μM L2 increased the cell viability to 70.1
9.8% (mean SD, n = 6, and P < 0.01). However, other
concentrations of L2, including 0.1, 10, and 100 μM, did not
improve the cell viability in HT-22 cells. In contrast, L4
pretreatment could protect cells from oxidative-stress-induced
cell damage in a dose-dependent manner. The HT-22 cell
viability increased significantly in the 1−100 μM L4 dose
range. More than 90% cell viability was restored with L4
pretreatment at 100 μM, the greatest protection observed in
the range studied. These cell viability assays are consistent with
cell toxicity studies, which indicated that L4 provided a
broader therapeutic window as well.
To investigate the mechanism of the cytoprotective effects of
L2 and L4, the expression level of the nuclear factor
(erythroid-derived-2)-like 2 (Nrf2) was examined by Western
blot. As a transcription factor, Nrf2 plays a critical role in the
regulation of a comprehensive and protective antioxidant
response. When properly activated, Nrf2 translocates into the
nucleus, where it binds to the antioxidant response element
and promotes the expression of antioxidant enzymes. Our
earlier work has shown that L2 could activate the Nrf2
pathway in the human blood−brain barrier CMEC/D3 cells.1
To investigate whether L4 also activates Nrf2 and how this
compared to L2, the HT-22 cells were treated for 24 h with 1
μM L2 and 100 μM L4 and the concentrations observed to
provide the highest protection against H2O2-induced cell death
for 24 h described in the studies above. Both L2 and L4
significantly increase the expression level of Nrf2 (Figure 8B).
Collectively, the data indicate that L2 and L4 protect neuronal
cells from oxidative-stress-induced cell damage. The neuro-
protective effects of L2 and L4 may mediate, at least in part,
through Nrf2 pathway activation as well.
Figure 7. (a) Cell survival versus logarithmic concentration of HT-22
cells dosed with cyclen, L1, L2, and L4 between 0.1 μM to 32 mM.
The data for cyclen, L1, and L2 are from ref 75.
L4 < L2< L1 < cyclen, suggesting that the bispyridol structure
is well-tolerated in biological models. As noted previously,
incorporation of the pyridol moiety modulates the basicity of
the ligand and pI. Therefore, it is interesting to note that the
Next, immortalized BV2 MGCs from C57BL/6J mouse
brain tissue were used to assess the ability of L4 compared to
Figure 8. Cytoprotective effects of L2 and L4 in HT-22 cells. (A) HT-22 cells pretreated with 0.1−100 μM L2 and 0.1−100 μM L4 for 24 h and
then incubated with 600 μM H2O2 for an additional 4 h. The cell viability was measured with WST-8 assay and represented by mean SD: (**)P
< 0.01 and (***)P < 0.001 compared with the H2O2-only group (n = 6). (B) Top panel: HT-22 cells treated with 1 μM L2 and 100 μM L4 for 24
h. Bottom panel: Relative pixel density of Nrf2 over loading control (β-actin): (*) P < 0.05 compared with the control (n = 3).
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L2 to provide protection from oxidative stress. MGCs work as
macrophages in the brain, and their activation occurs through
inflammation pathways in response to the development of Aβ
plaques.100,101 In vitro studies indicate that stimulated MGCs
phagocytose accumulated Aβ and increase levels of neuro-
toxins in the brain.102,103 Given this relationship, BV2 MGCs
were used as a model as well. A decrease in the cell viability to
44% was observed with the addition of H2O2 (Figure 9),
31% of L4 depleted over the time course of the experiment in
hepatocytes. The half-life for molecule L4 (>240 min) and
calculated Clint = 0.20 μL/min/million cells are consistent with
low clearance. The strong T1/2 and Clint values in both models
suggest that L4 will have slow clearance, thus increasing the
potential for delivery of the molecule to the blood−brain
barrier.
Cost Analysis. Finally, the cost of production is a
consistent concern among researchers in academia and
industry. Several high-profile news stories have highlighted
the exorbitant cost of pharmaceuticals.111,112 Therefore, we
carried out a Cost of Academic Methodology (CAM) analysis
for the synthesis of L4 as a means of understanding the
potential impact of production costs.113 In this analysis, the
CAM ($/mol) is equal to the sum of all prices ($/mol) for the
reagents and catalysts divided by the reaction yield. The
analysis does not include the cost of standard reagents (NaOH
or HCl, for example), workup, or labor, but this approach gives
a general sense of the overall comparative cost. The CAM for
L4 ($2926.81/mol) is 85% less than that of L2 thanks to the
lack of expensive catalysts required for the removal of
protecting groups (Figure S11). This analysis converts to an
estimated cost of $7.67 for each gram of L4 produced, which
would classify this as an inexpensive molecule.113
Figure 9. MTT cell survival assay of BV2 MGCs after exposure to L2
or L4 at concentrations of 0.0 μM to 750 nM and 0.533 mM H2O2 for
16 h. The final concentrations are shown. (****) Significance with
respect to the H2O2 control (n = 8) and P < 0.0001 (n = 8).
CONCLUSIONS
■
With the need for new molecules to be tested as therapeutics
for neurodegenerative diseases derived from oxidative stress, a
new bispyridol containing a tetraaza macrocycle was produced.
The success of the design of the water-soluble, new molecule
(L4) was evaluated by testing the antioxidant activity through
a series of assays and other studies. Radical quenching was
established using DPPH• assay, while halting the redox cycling
of copper(II) in the presence of oxygen and ascorbate was
evaluated using CCA assay. In both studies, L4 was shown to
outperform the parent pyridinophane L2. As suggested by
CCA assay, L4 binds copper(II) in a tetradentate fashion that
stabilizes the copper(II) species based on XRD and CV
studies. Furthermore, potentiometric data established that L4
has an ideal pI value of 7.3 for pharmaceuticals. The toxicity
and metabolic stability data in cellular models show remarkable
resistance to metabolism and low toxicity, thus further
encouraging more studies of L4 as a potential pharmacological
target to treat disorders associated with oxidative stress. Cell
studies in neuronal (HT-22) and MGC (BV2) cultures
indicate that L4, in particular, is a potent antioxidant and
inexpensive molecule that is capable of activating the Nrf2
pathway as an addition mechanism for protection against
oxidative stress. Altogether, this work shows how a simple
molecule can be designed using fundamental chemical
principles based on reactivity and coordination chemistry to
produce a highly functional potential therapeutic system that
will be explored in more biological contexts in future work.
similar to the response observed in HT-22 cells described
above. However, the cell viability was recovered (80−100%)
with femtomolar-to-nanomolar concentrations of both mole-
cules evaluated; no statistical differences between the L2 and
L4 data sets were observed, suggesting that both are effective at
preventing cell death from oxidative stress at very small
concentrations in the MGC model.
Metabolic Stability. The metabolic stability is a major
consideration when screening new drug entities for favorable
pharmacokinetic properties.104 It is a measurable parameter
that refers to the susceptibility of compounds to biotransfor-
mation. Both oral bioavailability and the plasma half-life of a
compound are closely tied to the metabolic stability of a
molecule. These factors impact the efficacy of the drug.
Therefore, L4 was evaluated using two in vitro assay
approaches to gauge applicability as a therapeutic.
The oxidation or reduction reactions facilitated by
cytochrome P450 and flavin monoxygenase enzymes are
described as phase I metabolism and can be modeled using
liver microsomes in the presence of an nicotinamide adenine
dinucleotide phosphate (NADPH)-regenerating system.105−107
In addition, intact hepatocytes are a model commonly used in
drug discovery and preclinical drug development to establish
the impact of drug-metabolizing enzymes related to four
categories of xenobiotic biotransformation: hydrolysis, reduc-
tion, oxidation, and conjugation, which encompass both phase
I and II metabolism.108 The method of substrate depletion was
used to determine the in vitro half-life.109 As illustrated in
Figure S9, molecule L4 exhibited high stability in microsomes
with a half-life greater than 120 min and 77% of molecule L4
was retained at the termination of the experiment. From this
data, L4 is classified as a low-intermediate clearance molecule
based on the intrinsic clearance (Clint) value of 5.40 μL/min/
mg.110 Likewise, exceptional stability was observed with only
EXPERIMENTAL METHODS
■
Caution! Perchlorate salts are explosive and should be handled with care;
such compounds should never be heated as solids. All chemical reagents
were purchased from either Millipore Sigma or Alfa Aesar and used
without further purification. Molecules L1−L3 and 1 were produced
using previously reported methods.67,78,99,114,115 The 12-membered
tetraaza macrocycles L1−L4 were isolated as hydrochloride salts prior
to metal-ion complexation, in accordance with standard practices.
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Elemental analyses were performed by Canadian Microanalytical
Services Ltd.
solid was washed with diethyl ether. L4 was isolated as an off-white
solid. Yield: 0.502 g (54%). 1H NMR (400 MHz, D2O, 25 °C): δ 6.54
(s, 4H), 4.38 (s, 8H). 13C NMR (101 MHz, D2O, 25 °C): δ 165.5,
151.3, 111.7, 52.1.66 Electronic absorption [H2O; λmax, nm (ε, M−1
cm−1)]: 255 (4400, sh). Elem anal. Found (calcd) for L4·3HCl·H2O·
0.5CH3OH: C, 41.51 (41.51); H, 5.64 (5.64); N, 12.38 (12.38).
Equilibrium Measurements. The reagents used in these studies
were of the highest analytical grade obtained from companies such as
Millipore, Sigma, Alfa Aesar, or Strem Chemicals Inc.
The pH-potentiometric titrations were carried out with a Metrohm
888 Titrando workstation, using a Metrohm 6.0234.100 combined
electrode. The titrated solutions (6.00 mL) were thermostated at 25
°C, the samples were stirred, and N2 was bubbled through the
solution to avoid the effect of CO2. Calibration of the electrode was
performed using a two-point calibration with potassium hydrogen
phthalate (pH = 4.008) and borax (pH = 9.177) buffers.
Physical Measurements. Electrospray ionization mass spectrom-
etry (ESI-MS) spectral analysis was performed on an Agilent 1200
series 6224 time-of-flight liquid chromatography/mass spectrometry
(LC−MS) spectrometer at 175 V (positive ESI scan) in water,
1
MeOH, CHCl3, or CH2Cl2. H and 13C NMR spectroscopies were
carried out in deuterated solvents at 25 °C. All spectra reported were
obtained on a Bruker Avance III (400 MHz) high-performance digital
NMR spectrometer. Electronic absorption spectra were collected
between 190 and 1100 nm using an Agilent 8453 UV−vis
spectrophotometer and a 3 mL quartz cuvette with a path length of
1.0 cm. Molar extinction coefficients were calculated utilizing the
Beer−Lambert law (A = εbc).
Synthesis of Tosylamide Monosodium Salt (TsNHNa).
TsNHNa was prepared according to literature procedures.62,64
Under reflux conditions, solid p-toluenesulfonamide (60.0 g, 0.35
mol) was added to sodium ethoxide (23.8 g, 0.35 mol) in absolute
ethanol (400 mL). This mixture was stirred under reflux conditions
for 2 h and then cooled. The insoluble TsNHNa salt was collected via
vacuum filtration and subsequently washed with absolute ethanol. The
isolated white solid (TsNHNa) was dried in vacuo to give >90% yield.
TsNHNa was used without further purification and can be stored
indefinitely.
Synthesis of Protected 8,17-Benzyloxy-4,13-bis(p-toluene-
sulfonyl)-1,4,11,13-tetraazabis(2,6-pyridinophane) (2).
TsNHNa (1.93 g, 10 mmol) was dissolved in 200 mL of DMF,
placed under N2, and heated to 80 °C. 1 (2.82 g, 10 mmol) was
dissolved in 50 mL of DMF and added dropwise to the above solution
via an addition funnel. Upon complete addition of TsNHNa, the
reaction mixture was stirred for 1 h. Next, a second 1 equiv of
TsNHNa (1.93 g, 10 mmol) was added (as a solid) to the reaction all
at once. The reaction was stirred for an additional 12 h at 80 °C under
N2. Upon reaction completion, the solvent was evaporated under
reduced pressure. Toluene was added to help form an azeotrope.
When about 5 mL of the solvent remained, an excess of cold MeOH
was added; 2 precipitated as an off-white powder. This mixture was
placed in a refrigerator for 12 h. Afterward, the mixture was vacuum-
filtered and the collected solid was washed with cold MeOH. 2 was
isolated as an off-white powder (3.11 g, 4 mmol, 40% yield).62−64
Crystals suitable for X-ray analysis were obtained via solvent diffusion
of DMF and MeOH.
The concentration of L4 was determined by pH-potentiometric
titration. In the pH-potentiometric titrations, 200−360 mL pH data
pairs were recorded in the pH range of 1.6−12.0. Calculation of [H+]
from the measured pH values was performed with use of the method
proposed by Irving and Miles116 by titrating a 0.02 M HCl solution (I
= 0.15 M NaCl) with a standardized NaOH solution (0.2 M). The
differences between the measured (pHread) and calculated pH (−log
[H+]) values were used to obtain the equilibrium H+ concentrations
from the pH data obtained in the titrations. The ion product of water
was determined from the same experiment in the pH range of 11.8−
12.0. The protonation constants were calculated from the titration
data with the PSEQUAD program.117
1
For the H NMR titrations, the adjustment of pD was carried out
with a Mettler Toledo SevenCompact pH/Ion S220 instrument
equipped with a Mettler Toledo InLab NMR pH electrode. −log
[H+] was measured directly in the NMR tube, after calibration of the
electrode with buffered aqueous solutions (pH 4.00, 7.00, and 10.00),
and the final pD was calculated by the equation pD = pH + 0.40.118 A
solution of L4 (c ≈ 0.02 M) was made in D2O, and the pD was
adjusted by adding 20% DCl in D2O or CO2-free 40% NaOD in D2O.
Sodium 2,2-dimethyl-2-silapentane-5-sulfonate was used as an internal
reference. UV absorption spectra for the spectrophotometric titrations
were collected between 200 and 400 nm using an Agilent Cary 60
UV−vis spectrophotometer and semimicro quartz cuvettes with a
path length of 1.0 cm. The pH of the L4 solution (c = 0.02 M) was
adjusted using solid NaOH and HCl gas.
Preparation of [Cu(L4)Cl]2Cl2 (CuL4). Molecule L4 (42.5 mg,
0.123 mmol) was weighed out and dissolved in 5 mL of water to give
a clear-light yellow solution. The pH of the ligand solution was
adjusted to 5.5 using a 1.0 M solution of KOH. Cu(ClO4)2·6H2O
(45.6 mg, 0.123 mmol) was subsequently weighed out and dissolved
in 1 mL of water. The metal solution was slowly added dropwise to
the pH-adjusted ligand solution to initiate metalation. Immediately
the solution color changed from light yellow to light blue; this
solution was stirred at room temperature, open to air, for 24 h. The
reaction mixture was subsequently evaporated under reduced pressure
to afford a light-green solid. The solid was dissolved in a minimum
amount of MeOH (1 mL), and a very small amount of water was
added (0.8 mL). Any remaining salts left undissolved were filtered out
of the solution; the filtered solution was set out for crystal growth.
After several days, more salts precipitated; these were subsequently
removed by filtration, and the solutions were set out for slow
evaporation and crystallization again. After several cycles of filtering
the solution and setting it out for slow evaporation, light-green X-ray-
quality crystals of CuL4 were isolated (15.1 mg, 0.017 mmol, 28%
yield). Electronic absorption [H2O; λmax, nm (ε, M−1 cm−1)]: 758
(23). Electronic absorption [DMF; λmax, nm (ε, M−1 cm−1)]: 873
(49). ESI-MS+. Calcd for [CuL4Cl]+ = [M]+ (Found): m/z 370.0258
(369.8742).
Synthesis of 1,4,11,13-Tetraazabis(2,6-pyridinophane)-
8,17-diol (L4). Lithium metal (1.3 g, 187 mmol) was cautiously
weighed out and cut into small pieces to expose unoxidized lithium
metal. These small pieces of lithium were added to a flask with 150
mL of dry tetrahydrofuran (THF). Naphthalene (3.0 g, 23 mmol) was
weighed out and added all at once to the flask. To produce the lithium
naphthalenide radical, the solution was stirred and then sonicated
until a dark-green color developed, indicating formation of the radical
species. The resulting dark-green solution was cooled to −78 °C in a
dry ice/acetone bath. 2 (2.6 g, 3.4 mmol) was dissolved in 100 mL of
THF to form a brown slurry. This slurry was added dropwise to the
lithium naphthalenide solution in the flask. After complete addition of
2, the reaction mixture was allowed to slowly warm to room
temperature. The reaction was then stirred at room temperature for
12 h. Afterward, the reaction mixture was placed on ice and
hydrolyzed with water, a precipitate developed, and the solution had a
light-brown color. Upon quenching with water, 6 M HCl and then 1
M HCl was added to the reaction until there was no longer a
discernible layer between the water and THF, and no additional color
change was observed. This aqueous solution was washed several times
with diethyl ether. After extraction with diethyl ether, the aqueous
layer was evaporated under reduced pressure until about 15 mL of
solution was present. The remaining yellow solution was transferred
to a falcon tube and evaporated using a lyophilizer. The resulting
semidry solid was dissolved in a minimal amount of MeOH. To this
solution were added copious amounts of ether and additional MeOH
until an off-white solid precipitated. This mixture was left to stir for
several hours. The solution was vacuum-filtered, and the collected
X-ray Crystallography. A Leica MZ 75 microscope was used to
identify samples suitable for analysis. A Bruker APEX-II CCD
diffractometer was employed for crystal screening, unit cell
determination, and data collection, which was obtained at 100 K.
The Bruker D8 goniometer was controlled using the APEX3 software
H
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suite, version 1.119 The samples were optically centered with the aid
of a video camera, so that no translations were observed as the crystal
was rotated through all positions. The X-ray radiation employed was
generated from a Mo Kα sealed X-ray tube (λ = 0.71076) with a
potential of 50 kV and a current of 30 mA and fitted with a graphite
monochromator in the parallel mode (175 mm collimator with 0.5
mm pinholes).
into 7-hydroxy-CCA (λex = 395 nm and λem = 450 nm). The general
order of addition is as follows: PBS buffer (2200 μL for positive
control, 2150 μL for 0.5 equiv samples, 2100 μL for 1 equiv samples,
and 2300 μL for negative controls), CCA (100 μM, 500 μL), Desferal
(1 μM, 100 μL),95,125 molecule L2 or L4 (1/2 equiv = 5 μM, 50 μL; 1
equiv = 10 μM, 100 μL), copper(II) (10 μM, 100 μL), and then
ascorbate (300 μM, 100 μL).
Cell Culture Methods. HT-22 Cell Culture. The mouse neuronal
hippocampal cell line (HT-22) were obtained from the UNT Health
Science Center. HT-22 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM), supplemented with 100 units/mL
penicillin/100 μg/mL, streptomycin, 1 mM L-glutamine (100 mM,
5 mL), and charcoal-stripped fetal bovine serum (FBS; 15%, Sigma-
Aldrich). Both cell lines were grown in a water-jacketed incubator at
37 °C with 5% CO2 and 95% air atmospheric conditions. To prepare
dilutions of compounds, the same media were used excluding the
addition of the serum and antibiotics.
Cell Viability in HT-22. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenylte-
trazolium bromide (MTT) assay was used to determine the cell-line
sensitivity to the drugs used in this study. To perform this assay, 5 ×
103 cells/well were plated into 96-well plates for 24 h and then treated
with increasing concentrations of the molecule of interest in replicates
of 8 and incubated for 16 h at 37 °C. The medium was replaced by
100 μL of 1 mg/mL MTT in a serum-free medium. The cells were
then incubated for 4 h at 37 °C, after which MTT was replaced by
100 μL of dimethyl sulfoxide (DMSO) to solubilize the precipitate.
The plate was placed in a shaker for 5 min, and the absorbance was
measured at 540 nm using a plate reader (Molecular Dynamics). The
data obtained were processed using GraphPad Prism 5 for Windows
to determine the EC50 values presented. Analysis tables are available
in the Supporting Information.
Cell Viability Assay for H2O2 Studies. The cell viability was
measured by a colorimetric cell viability kit (PK-CA705-CK04;
Promokine, Heidelberg, Germany) with the tetrazolium salt WST-8
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophen-
yl)-2H-tetrazolium, monosodium salt], which can be bioreduced to a
water-soluble orange formazan dye by dehydrogenases present in the
viable cells. The amount of formazan produced is directly propor-
tional to the number of living cells. Cells were incubated with or
without L2 and L4 for 24 h and then treated with 600 μM H2O2 for 4
h. After treatment, 10 μL of the WST-8 solution was added to each
well of the culture plate and incubated for 2 h in the incubator. The
absorption was evaluated at 450 nm using a microplate reader
(BioTek, Winooski, VT).
Western Blot Analysis. The protein concentration was determined
with a BCA assay kit (23225; Thermo Scientific, Rockford, IL). Equal
amounts of protein were boiled in a Laemmli buffer (Bio-Rad,
Richmond, CA), loaded onto 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis gel, and transferred to a 0.2 μm
poly(vinylidene difluoride) membrane (GE Healthcare, Boulder,
CO). The membranes were incubated with the appropriate primary
antibodies overnight at 4 °C and then incubated with the appropriate
secondary antibodies for 1 h. Detection was performed using an ECL
Western blotting detection system (Thermo Scientific, Rockford, IL).
The immunoblot was analyzed with a Bio-Rad imaging system
(Versadoc 5000 MP Imaging System, Bio-Rad, Richmond, CA).
MGCs. The BV2 MGCs were a gift from Dr. Meharvan Singh. The
BV2 cells were incubated at 37 °C in 5% CO2. Cells were cultured
and passaged in 10 cm tissue culture dishes in DMEM, supplemented
with 5% L-glutamine, 5% penicillin−streptomycin, and 15% FBS. The
BV2 cells were passaged when plates reached 80−90% confluence. A
serum-free medium was used for dilutions of compounds.
MTT assay was utilized to measure the cell viability with
pretreatment of a L2 or L4 compound, administered 1 h before the
induction of oxidative stress. The BV2 cells were first plated in a 96-
well plate at a density of 5000 cells/100 μL of the complete medium
per well. After 24 h, the antioxidant molecule was added to each well
to provide final concentrations of 416 nM to 41.6 fM. Each
concentration was replicated in six wells per column, including a
control column that did not receive treatment. Following treatment,
CuL4 (CCDC 1910897) Structure Determination. A trans-
lucent light-green blocklike crystal of CuL4 (0.279 × 0.160 × 0.126
mm) was mounted on a 150 μm cryoloop and used for X-ray
crystallographic analysis. The crystal-to-detector distance was set to
50 mm, and the exposure time was 10 s/deg for all data sets at a scan
width of 0.5°. A total of 2180 frames were collected, and the data
collection was 99% complete. The frames were integrated with the
Bruker SAINT software package120 using a narrow-frame algorithm.
Integration of the data used a monoclinic unit cell, yielding a total of
45954 reflections to a maximum θ angle of 30.13° (0.71 Å
resolution), of which 5217 reflections were independent with Rint
=
4.99%. The data were corrected for absorption effects using the
multiscan method SADABS.121 Using Olex2,122 the structure was
solved with the SHELXS123 structure solution program using direct
methods and refined with the SHELXL124 refinement package using
least-squares minimization. All H and non-H atoms were refined using
anisotropic thermal parameters. The thermal ellipsoid molecular plots
(50%) were produced using Olex2.122
Electrochemical Measurements. CV was carried out with an
EC Epsilon potentiostat (C-3 cell stand) purchased from BASi
Analytical Instruments (West Lafayette, IN). A glassy carbon (GC)
electrode from BASi (MF-2012), 3 mm in diameter, was polished on
a white nylon pad (BASi MF-2058) with different-sized diamond
polishes (15, 6, and 1 μm) to ensure a mirrorlike finish. Between each
measurement, the GC electrode was polished with three diamond
polishes. A three-electrode cell configuration was used with GC as the
working electrode, a silver wire (0.5 mm diameter) quasi-reference
electrode housed in a glass tube (7.5 cm × 5.7 mm) with a porous
CoralPor tip, and a platinum wire (7.5 cm) as the counter electrode
(BASi MW-1032). All solutions were bubbled with N2 gas for at least
15 min prior to experimentation and kept under a humidified N2 gas
blanket. All potentials herein are reported versus Fc/Fc+ (E1/2 = 0.0
V). For each electrochemical analysis, 3.0 mg of complex was
dissolved in 3.0 mL of anhydrous DMF containing 0.1 M
tetrabutylammonium perchlorate as the supporting electrolyte.
DPPH• Assay. A stock solution of DPPH• was prepared by
dissolving 17.8 mg in 300 mL of MeOH (150 μM). A solid sample of
L4 or BHT was dissolved in 10 mL of MeOH (stock concentration 5
mM). A solid sample of L2 was dissolved in 10 mL of water, and the
pH was adjusted to 7 using 1.0 M KOH. The water was then
removed, and the sample was dissolved in 10 mL of MeOH (stock
concentration 5 mM). Next, the sample solutions were prepared in 7
mL vials by diluting the stock solutions (5 mM) with MeOH to
various concentrations (2−3000 μM, 1 mL). Next, each working
sample was prepared by mixing 100 μL of one of the prepared
samples with 100 μL of the DPPH stock directly in the 96-well plate
to a final concentration ranging from 1 to 1500 μM. A separate well
plate was used for each sample, and each concentration was analyzed
in triplicate. An aliquot of the DPPH stock solution (100 μL) was
mixed with MeOH (100 μL) and used as a negative control for each
sample. The samples were incubated in the dark for 30 min at room
temperature. For analysis, each well plate was run using the BMG
Labtech FLUOstar Omega UV/vis absorbance spectrophotometer
microplate reader, and the absorbance at 516 nm of each sample was
measured. Each experiment was performed in triplicate (n = 3). The
final absorbance values have been normalized to the average (n = 3)
absorbance of the negative control DPPH sample and are expressed as
the percentage of DPPH• quenched.
CCA Assay. All solutions were prepared in a phosphate-buffered
saline (PBS) buffer (1X, pH 7.4) except CuSO4·6H2O, which was
dissolved and diluted in Milli-Q water. The final sample volume was 3
mL. Each experiment was performed in triplicate (n = 3). Hydroxyl
radical production was followed by measuring the conversion of CCA
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the plate was maintained in an incubator for 1 h. To provoke oxidative
stress, all cells were then treated with H2O2 at a final concentration of
0.533 mM/well and the plate was then incubated for 16 h.
Supernatants were removed, and 100 μL of MTT (1 mg/mL
serum-free medium) was added to all wells and incubated for 4 h.
Subsequently, supernatants were removed, 100 μL of DMSO was
added to each well, and the plate was placed on a rocker for 5 min.
The absorbance was then measured using a plate reader (BMG
LabTech FLUOstar Omega) at an absorbance of 540 nm. The data
presented were standardized to cells receiving no treatment (100%)
and the results of an average of three plates for each compound
studied. The data were analyzed with GraphPad Prism 5 for Windows.
Mouse Microsome Stability (Phase I). Male ICR/CD-1 mouse
microsomes (Lot ZMC) were purchased from BioIVT (Baltimore,
MD). A total 0.25 mg of microsomes was added on ice to a 50 mM
Tris, pH 7.5, solution, containing L4 added from a DMSO stock. The
final concentration of the compound after the addition of all reagents
was 2 μM. An NADPH-regenerating system [1.7 mg/mL NADP, 7.8
mg/mL glucose-6-phosphate, 6 U/mL glucose-6-phosphate dehydro-
genase in 2% (w/v) NaHCO3/10 mm MgCl2] was added for analysis
of the phase I metabolism. The tube was then placed in a 37 °C
shaking water bath. At various time points after the addition of phase I
cofactors, the reaction was stopped by the addition of 0.5 mL of
MeOH containing an internal standard (IS), tolbutamide, and 0.2%
formic acid to quench the reaction and precipitate protein. Time 0
samples were quenched prior to the addition of the compound or
NADPH-regenerating system. The samples were incubated 10 min at
room temperature and then spun at 16100g for 5 min in a
microcentrifuge. The supernatant was analyzed by LC−MS/MS.
Analytical methods were developed for each compound using a Sciex
4000 QTRAP (Foster City, CA), a combination triple quadrupole/
ion-trap instrument. The parent ion and two most prominent
daughter ions were followed to confirm the compound identity,
although only the most abundant daughter was used for quantitation.
L4 was monitored by the 273.1 to 137.0 transition and the IS
tolbutamide by the 269.1 to 169.9 transition using the following ion
source parameters: CUR = 25, CAD = high, IS = 4000, TEM = 600,
GS1 = 70, and GS2 = 70. A Shimadzu Prominence liquid
chromatograph (Columbia, MD) with a Phenomenex Luna C8 (5
μm packing; 100 × 4.6 mm; Torrance, CA) was used for
chromatography under the following conditions: buffer A, water +
0.1% formic acid; buffer B, MeOH + 0.1% formic acid; flow rate, 0.7
mL/min; 0−2 min gradient to 1% B, 2.0−3.0 min gradient to 95% B,
3.0−4.0 min gradient to 95% B, and 4.0−4.1 min gradient to 1% B
and 4.1−5.0 1% B.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.9b02932.
■
S
Reaction schemes, X-ray crystallographic data for 2 and
CuL4, potentiometric data, and assay data (PDF)
Accession Codes
CCDC 1910897 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Author
*Email: kayla.green@tcu.edu.
ORCID
■
́
Gyula Tircso: 0000-0002-7896-7890
Kayla N. Green: 0000-0001-8816-7646
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by the National Institutes of Health
(Grant R15GM123463 to K.N.G., G.A., and M.C.). The
authors are also grateful for generous financial support from
TCU Andrews Institute of Mathematics & Science Education
(to K.N.G.), Cambridge Isotope Laboratories, and a TCU
Research and Creativity Activity Grant (to K.N.G.). Metabolic
stability studies were conducted by the Preclinical Pharmacol-
ogy Core at the University of Texas Southwestern Medical
Center. Gy.T. is grateful for support from the Hungarian
National Research, Development and Innovation Office
(Project NKFIH K-120224), and for a Janos Bolyai Research
Scholarship of the Hungarian Academy of Sciences. Gy.T.
thankful for a scholarship supported by the UNKP-18-4 New
National Excellence Program of the Ministry of Human
Capacities. The research was also supported, in part, by the
European Union and cofinanced by the European Regional
Development Fund under Projects GINOP-2.3.2-15-2016-
00008 and GINOP-2.3.3-15-2016-00004 to Gy.T. The Table
of Contents background picture is by Jason Sells of College
Station, TX, and was used with his permission.
́
Mouse Hepatocyte Stability (Phases I and II). L4 (DMSO stock, 2
μM final concentration) was incubated with murine hepatocytes (Lot
NNS) for 0−240 min at 37 °C and 5% CO2. The final concentration
of hepatocytes in the reaction was 1 × 106/mL. The enzymatic
reaction was quenched with a 2-fold volume of MeOH containing
0.15% formic acid and the IS tolbutamide. Samples were vortexed for
15 s, incubated at room temperature for 10 min, and spun for 5 min at
16100g. The supernatant was collected and analyzed by LC−MS/MS,
as described above for the microsome stability assay.
́
The method described by McNaney et al.109 was used with
modification for determination of the metabolic stability half-life by
substrate depletion. A “% remaining” value was used to assess the
metabolic stability of a compound over time. The LC−MS/MS peak
area of the incubated sample at each time point was divided by the
LC−MS/MS peak area of the time 0 (T0) sample and multiplied by
100. The natural logarithm (ln) of the % remaining of the compound
was then plotted versus time (in minutes), and a linear regression
curve was plotted going through the y intercept at ln(100). If the
metabolism of a compound failed to show linear kinetics at a later
time point, those time points were excluded. The half-life (T1/2) was
calculated as T1/2 = 0.693/slope. To determine the intrinsic clearance,
the ln peak area ratio (compound peak area/IS peak area) was plotted
against time and the gradient of the line determined.126−129
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