A Novel Iron(II) Preferring Dopamine Agonist Chelator as Potential Symptomatic and Neuroprotective Therapeutic Agent for Parkinson's Disease

Article abstract of DOI:10.1021/acschemneuro.6b00356

Parkinson's disease (PD) is a progressive neurodegenerative disorder, and development of disease-modifying treatment is still an unmet medical need. Considering the implication of free iron(II) in PD, we report here the design and characterization of a novel hybrid iron chelator, (-)-12 (D-607) as a multitarget-directed ligand against PD. Binding and functional assays at dopamine D₂/D₃ receptors indicate potent agonist activity of (-)-12. The molecule displayed an efficient preferential iron(II) chelation properties along with potent in vivo activity in a reserpinized PD animal model. The compound also rescued PC12 cells from toxicity induced by iron delivered intracellularly in a dose-dependent manner. However, Fe³⁺ selective dopamine agonist 1 and a well-known antiparkinsonian drug pramipexole produced little to no neuroprotection effect under the same experimental condition. These observations strongly suggest that (-)-12 should be a promising multifunctional lead molecule for a viable symptomatic and disease modifying therapy of PD.

Full text of DOI:10.1021/acschemneuro.6b00356

Letter
pubs.acs.org/chemneuro
A Novel Iron(II) Preferring Dopamine Agonist Chelator as Potential
Symptomatic and Neuroprotective Therapeutic Agent for
Parkinson’s Disease
Banibrata Das,† Ashoka Kandegedara,† Liping Xu,† Tamara Antonio,‡ Timothy Stemmler,†
Maarten E. A. Reith,‡ and Aloke K. Dutta*,†
†Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48202, United States
‡Department of Psychiatry, New York University, New York, New York 10016, United States
S
* Supporting Information
ABSTRACT: Parkinson’s disease (PD) is a progressive neurodegenerative disorder, and development of disease-modifying
treatment is still an unmet medical need. Considering the implication of free iron(II) in PD, we report here the design and
characterization of a novel hybrid iron chelator, (−)-12 (D-607) as a multitarget-directed ligand against PD. Binding and
functional assays at dopamine D2/D3 receptors indicate potent agonist activity of (−)-12. The molecule displayed an efficient
preferential iron(II) chelation properties along with potent in vivo activity in a reserpinized PD animal model. The compound
also rescued PC12 cells from toxicity induced by iron delivered intracellularly in a dose-dependent manner. However, Fe3+
selective dopamine agonist 1 and a well-known antiparkinsonian drug pramipexole produced little to no neuroprotection effect
under the same experimental condition. These observations strongly suggest that (−)-12 should be a promising multifunctional
lead molecule for a viable symptomatic and disease modifying therapy of PD.
KEYWORDS: Parkinson’s disease, D2/D3 agonist, iron chelation, neuroprotection, multifunctional drug
attractive therapeutic strategy to be used against the pro-
gression of PD.14
arkinson’s disease (PD) is a major neurodegenerative
Pdisorder affecting 1−2% of the elderly population, causing
profound motor impairments that include tremors at rest,
rigidity, bradykinesia, and postural instability along with non-
motor symptoms such as autonomic, cognitive, and psychiatric
problems.1−3 It is believed that environmental factors converging
on oxidative stress, mitochondrial dysfunction, inflammation, and
aberrant protein aggregation account for most cases of PD.4,5
Nigral iron elevation in early stages of PD progression is a
consistently reported feature of the disease,6−8 which is also
confirmed by magnetic resonance imaging9 and ultrasound
studies.10 The proposed pathogenic mechanisms for iron-
mediated PD degeneration involve increased oxidative stress,
owing to the interaction between intracellular ferrous iron and
hydrogen peroxide (H2O2) in the Fenton reaction to generate
hydroxyl free radicals,11 and may also cause aggregation of
α-synuclein (α-Syn) protein to form toxic oligomers.12,13
Therefore, the critical cross talk between iron accumulation
and neurodegeneration suggests that iron chelation might be an
Currently, the mainstay treatment of PD focuses on the
restoration of dopamine (DA) activity within the nigrostriatal
tract either with DA agonists or with L-dopa, thereby alleviating
only the symptoms without affecting the course of the disease
progression.15 L-Dopa usage is unfortunately associated with
side effects including dyskinesia, and its long-term use can
produce sudden “on−off” effects.16 Moreover, no neuroprotective
drugs have been identified or approved by the United States
Food and Drug Administration (FDA) so far for the treat-
ment of PD,17,18 thereby necessitating research for one. In
this regard, multifunctional drugs have now been increasingly
implicated in disease-modifying treatment of PD and in the
neurodegeneration in general.19,20 Our approach in developing
such agents involves alleviating motor dysfunction in PD, along
Received: October 19, 2016
Accepted: January 20, 2017
Published: January 20, 2017
© XXXX American Chemical Society
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with slowing or halting the neurodegeneration process by our
multifunctional drug development approach.21−26
generate reactive oxygen species.37 Thus, a Fe2+ preferring
chelator should provide greater beneficial effect against neuro-
degeneration. The lead molecule in question in this study,
(−)-12 (D-607) (Figure 1), not only exhibits potent in vivo
activity in a PD animal model, but also displays activities
pertinent to neuroprotection, such as preferential complexation
with Fe2+ and inhibition of iron-induced neuronal cell death.
Such a drug should provide not only symptomatic benefit but
also a degree of neuroprotection over the course of treatment;
the latter would endow the compound with disease-modifying
capability.
Several iron chelators have been evaluated in the preclinical
and clinical studies of PD.27,28 An iron chelator, deferiprone,
has shown promising results in a recently completed 12-month
clinical trial in early stage PD patients,27 indicating real
potential application of suitable iron chelators in disease
modifying therapy. Also, the drug clioquinol, which exhibited
neuroprotection in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine)29 and efficacy in a transgenic α-Syn animal
models,30 is a chelator of oxidized form of iron (Fe3+) and not a
multifunctional drug. We previously demonstrated the develop-
ment of iron chelating DA agonist, 1 (Figure 1), which formed
complexes with Fe3+.23 As part of our continued endeavor to
develop neuroprotective and disease modifying drug for PD, we
report here the design and synthesis of novel, multifunctional
compounds based on our hybrid drug design template,31
in which D2/D3 agonist headgroup is attached to a bipyridyl
moiety, a known iron chelating agent having preferential affinity
for Fe2+.32−35 The rationale for employing the bipyridyl frag-
ment is based on the fact that the ratio of Fe3+:Fe2+ from 2:1 in
normal subjects is shifted to 1:2 in PD patients.36 Moreover,
the chelatable iron in the brain intracellular compartment exists
in the Fe2+ state, which participates in the Fenton reaction to
RESULTS AND DISCUSSION
■
Our attempt to find a multifunctional compound was based on
the intracellular Fe2+ chelator 2,2′-bipyridine as a potential
disease modifying agent in PD, and accordingly we designed
and synthesized our novel neuroprotective iron chelator (−)-12
(Scheme 1), in which the bipyridyl fragment was appended in
our core hybrid structure. The starting compound 3 was
obtained by alkylating piperazine with (2-bromoethoxy)-tbutyl-
dimethylsilane (2) in the presence of K2CO3 under reflux.
A palladium-catalyzed Stille coupling reaction between 5,
prepared according to a procedure described previously,38 and
2,5-dibromopyridine yielded intermediate 6. The N-alkylation
Figure 1. Structures of iron chelators, dopamine agonists, and multifunctional iron chelating dopamine agonists.
a
Scheme 1. Synthetic Scheme for the Target Compounds
a
Reagents and conditions: (a) K2CO3, CH3CN, reflux, overnight; (b) tri-n-butyltin chloride, n-BuLi, Et2O, −78 °C to rt, 18 h; (c) 2,5-dibromopyridine,
Pd(PPh3)4, xylene, reflux, 24 h; (d) 3, Pd(OAc)2, BINAP, Cs2CO3, toluene, reflux, 12 h; (e) n-Bu4NF, THF, 0 °C to rt, 2 h; (f) SO3·py,
CH2Cl2/DMSO (2:1), Et3N, 0 °C to rt, 1.5 h; (g) 10, NaBH(OAc)3, CH2Cl2, rt, 48 h; (h) 11, NaBH(OAc)3, CH2Cl2, rt, 48 h; (i) 48% aqueous HBr,
reflux, 5 h.
B
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was performed by refluxing the mixture of 3, 6, cesium
carbonate, BINAP (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl),
and palladium(II) acetate in toluene to give 7 in excellent yield.
The silyl group of compound 7 was removed by treatment with
n-Bu4NF (tetrabutylammonium fluoride) in THF to afford
alcohol 8. Subsequent oxidation of the hydroxyl group using
pyridine-sulfur trioxide furnished the corresponding aldehyde 9,
which was used without further purification to reductively
alkylate the secondary amines (−)-1021,39 and (−)-1140 in the
presence of NaBH(OAc)3 (sodium triacetoxyborohydride) to
furnish the test compounds (−)-12 and (−)-13. It is worth
mentioning here that 8 can be oxidized using both Swern and
Parikh−Doering oxidation conditions, but we used the later
one for milder reaction condition. Finally, demethylation of
(−)-13 was accomplished by refluxing with 48% aqueous HBr
to give (−)-14 as HBr salt. Enantiopure compounds (−)-12
and (−)-13 were also converted to their corresponding
HCl salts by treatment with ethereal HCl. All the final com-
pounds were characterized by 1H NMR, 13C NMR, HRMS, and
elemental analysis (refer to the Supporting Information for
details).
(−)-14 revealed very high affinity at both D2 and D3 receptors
(Ki, D2 = 7.63 nM, D3 = 1.00 nM, D2/D3 = 7.6) indicating that
the 5-hydroxy aminotetralin agonist moiety shows relatively
higher affinity toward D2 than D3 receptors compared to the
2-aminothiazole group.41
Following binding analysis, agonist activities of the optically
active compounds for human DA D2 and D3 receptors expressed
in chinese hamster ovary (CHO) cells were measured by moni-
toring stimulation of [35S]GTPγS (guanosine 5′-O-[γ-thio]-
triphosphate) binding in comparison to stimulation by the full
agonist DA.21 As shown in Table 2 and Figure S4B in the
Supporting Information, only (−)-12 demonstrated high
affinity and full agonist activity at both D2 and D3 receptors
(Emax close to 100%) while (−)-14 was found to be a partial
agonist (Emax = 63−68%) and efficacious in stimulating both
receptors (EC50 (GTPγS); D2 = 3.14 and D3 = 0.62 nM). None
of the compounds displayed appreciable selectivity for D3 vs D2.
It should be noted that both (−)-13 and (−)-14 revealed
partial agonist activities at D2/D3 receptors as did their parent
reference molecule 5-OH-DPAT. It is also quite interesting to
find a difference between binding (Ki) and intrinsic activity
(EC50) data for compound (−)-12; however, at present, we do
not have a full explanation for this phenomenon. One
possibility is that the compound might be acting as an agonist
mostly allosterically while exhibiting weak interaction at the
orthosteric binding sites. Thus, our binding and functional
assay results indicated that compounds with a 2,2′-bipyridine
moiety retain not only high affinity for binding to D2/D3
receptors but also appreciable agonist activity at both receptors.
Our goal is to identify compound with full agonist activity to
provide symptomatic beneficial effect in PD. Therefore, based
on full agonist activity, we decided to pursue compound (−)-12
for further characterization.
To evaluate receptor binding of the final compounds, a
radioligand competition assay was conducted and the binding
affinity profiles were compared with that of the reference agent
(−)-5-OH-DPAT (5-hydroxy-N,N-dipropyl-2-aminotetralin)
(Table 1).21 Binding affinity was determined by inhibition of
Table 1. Inhibition Constants Determined by Competition
Experiments Assessing [3H]Spiroperidol Binding to Cloned
a
D2L and D3 Receptors Expressed in HEK-293 Cells
Ki (nM)
compd
D2L, [3H]spiroperidol D3, [3H]spiroperidol D2L/D3
Spectral feature perturbations in the UV−vis spectra during
Fe(II) or Fe(III) addition to the ligand at different ratios were
used to measure iron oxidation state specific binding constants
as well as metal to ligand ratios for metal binding to compound
(−)-12 (Figure S6 in the Supporting Information). All samples
were prepared, and spectra were collected, anaerobically to
stabilize the reduced form of iron and to keep solutions con-
ditions constant. Apo compound (−)-12 has UV−vis absorbance
feature maxima at 260, 308, and 364 nm (Figure 2A); holo
ligand has maxima values at 330 and 510 nm (Figure 2B and C
for Fe(III) and Fe(II), respectively). Progressive loading of
Fe(II) onto (−)-12 showed an exponential increase in the
spectral profile at A510 vs [Fe(II)]/[ligand] (Figure 2, inset
(c)). This A510 spectral profile could easily be fit using eq 1
within Dynafit42 for a 1:3 Fe(II) vs ligand stoichiometric
profile. In eq 1, L is the compound (−)-12 and Kd1 is the
(−)-5-OH-DPAT
(−)-12
(−)-13
153 32
674 17
227 4.0
7.63 1.78
2.07 0.38
13.4 1.0
8.90 2.04
1.00 0.34
74
50
25
(−)-14
7.6
a
Results are the mean SEM of 3−6 experiments, each performed in
triplicate.
[3H]spiroperidol binding to rat dopamine D2 and D3 receptors
expressed in human embryonic kidney-293 (HEK-293) cells.
As shown in Table 1, compound (−)-12 exhibited moderate
affinity for both D2 and D3 receptors (Ki, D2 = 674 nM,
D3 = 13.4 nM) along with moderate selectivity at the D3
receptor compared to the D2 receptor (D2/D3 = 50) (see also
Figure S4A in the Supporting Information). A similar profile
was also observed for compound (−)-13 (Ki, D2 = 227 nM,
D3 = 8.90 nM, D2/D3 = 25). On the other hand, compound
a
Table 2. Stimulation of [35S]GTPγS Binding to Cloned Human D2 and D3 Receptors Expressed in CHO Cells
hCHO−D2
[35S]GTPγS, EC50 (nM)
376 39
41
hCHO−D3
[35S]GTPγS, EC50 (nM)
compd
Emax (%)
Emax (%)
D2/D3
DA
100
7.26 0.21
0.63 0.08
13.5 4.2
19.1 4.6
0.62 0.18
100
52
(−)-5-OH-DPAT
(−)-12
(−)-13
6
80
4
6
75
4
65
51.6 2.4
119 21
101
83.4 0.9
33.2 4.2
67.9 2.4
3.8
6.2
5.06
29.0 2.3
62.9 4.9
(−)-14
3.14 0.92
a
EC50 is the concentration producing half maximal stimulation. For each compound, maximal stimulation (Emax) is expressed as a percent of the Emax
observed with 1 mM (D2) or 100 μM (D3) of the full agonist DA (Emax, %). Results are the mean SEM for 3−6 experiments, each performed in
triplicate.
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calculated Kd of 1.60 ( 0.33) × 10−13 M for Fe(II) binding to
compound (−)-12.
Kd1
Fe(II) − LLL ←→ Fe(II) + L + L + L
(1)
Progressive loading of Fe(III) onto (−)-12 showed a slower
increase in the spectral profile at A510 vs [Fe(III)] (Figure 2,
inset (b)) at the same metal/ligand ratios. The A510 increase
profiles were fit using eq 2 with a 1:1 Fe(III)/ligand stoichi-
ometry where L is again (−)-12 and Kd2 is the dissociation
constant for the Fe(III)/ligand 1:1 complexation reaction.
Simulations of the representative data in Figure 2, inset b
resulted in a calculated Kd of 2.38 ( 0.59) × 10−4 M for Fe(III)
binding to compound (−)-12. Our experiment thus revealed
exceptionally high preferential binding of (−)-12 to Fe(II)
compared to Fe(III), which further corroborates with the
literature publications.33−35
Figure 2. UV−vis spectra of ligand and ligand to Fe(II) and Fe(III)
complexes used for binding affinity analysis. Absorbance of apo (−)-12
(A), the ligand complex with Fe(III) (B), and the complex with Fe(II)
(C) are shown. Arrows denote the direction of the absorbance changes
with increasing concentrations of iron. Inset: Absorbance changes at
510 nm as a function of [Fe(III)] and [Fe(II)] are shown in (b) and
(c), respectively.
Kd2
Fe(III) − L ←→ Fe(III) + L
(2)
The neuroprotective effect of (−)-12 was next explored in
neuronal PC12 cells against iron-induced neuronal cell death.
Previous studies have confirmed that Fe(III) complexed to the
lipophilic ligand 8-hydroxyquinoline (8HQ) effectively transfers
reactive metal (free iron) into and across the cells.43,44 Once
Fe(III) is internalized into the cells, it can be reduced to Fe(II)
dissociation constant for the Fe(II) complexation reaction. Simu-
lations of the representative data in Figure 2, inset c resulted in a
Figure 3. Dose-dependent effect of iron, 8-hydroxyquinoline and (−)-12 on cell viability of PC12 cells. (a) PC12 cells were treated with different
doses of iron followed by incubation for 24 h. (b) Similarly, PC12 cells were first treated with varying concentration of 8-HQ for 30 min and then
incubated for 24 h with fresh culture medium. (c) PC12 cells were treated with different doses of (−)-12 for 24 h. Values shown are means SDs of
three independent experiments performed in four to six replicates. One-way ANOVA analysis (F(8, 63) = 253, p < 0.0001 for (a), F(8, 63) = 82.98,
p < 0.0001 for (b), and F(8, 62) = 9.89, p < 0.0001 for (c). ANOVA was followed by Tukey’s multiple comparison post hoc tests (#p < 0.05, ##p <
0.01, ###p < 0.001, and ####p < 0.0001 compared to the control groups).
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probably by two iron reducing systems, an ascorbate−dependent
and a NADPH-dependent systems,45 and this Fe(II) then
reacts with H2O2 in the Fenton reaction to generate hydroxyl
radical. Based on this mechanism, it was shown that Fe(III)-
8HQ complex is neurotoxic in the cerebral endothelial cells by
augmenting oxidative stress through hydroxyl radical produc-
tion and lipid peroxidation;44,46,47 however, such a phenom-
enon has not been demonstrated in neuronal PC12 cells, and,
in particular, this paradigm has not been applied to assess the
neuroprotective potential of an agent in PD.
Here we show that treatment of PC12 cells with increasing
concentrations of (−)-12 alone (0.1−10 μM) showed no sig-
nificant cell loss compared to untreated control (Figure 3c),
indicating the nontoxic profile of the compound at the doses
tested. Moreover, iron or 8HQ alone only conferred appreciable
toxicity at a higher concentration range (Figure 3a and b,
respectively). On the other hand, cells treated with Fe-8HQ
complex in a ratio of 1:2 for 30 min results in a significant dose-
dependent neurotoxicity; 35% cell death was observed with a
concentration of 20/40 μM (Fe/8HQ) in the complex solution,
and this concentration was used in subsequent in vitro experi-
ments (Figure 4a). This indicates 8HQ mediated transport of
Fe(III) into the PC12 cells and intracellular conversion of
Fe(III) to Fe(II) to produce oxidative stress and cell death.
Figure 4b shows a dose-dependent protection effect of (−)-12
against iron-induced cell death. Thus, when the cells were
pretreated with the iron chelator for 1 h followed by Fe/8HQ
(20/40 μM) treatment for 30 min, the compound dose-
dependently protected the cells from the neurotoxic insult and
the greatest protective effect was obtained at concentration of
20 μM which increased the cell survival by 20% compared to
Fe/8HQ (20/40 μM) treated alone. Furthermore, we have
carried out additional neuroprotection experiments with two
higher toxic doses (30/60 and 40/80 μM) of Fe(III)-8HQ
complex. These doses by themselves produced 54% (30/60
μM) and 80% (40/80 μM) toxicity, respectively. As shown in
Figure 5a for 30/60 μM dose and also in the supplemental
section for 40/80 μM dose (Figure S8), compound (−)-12 was
able to provide significant neuroprotection dose dependently in
both cases, indicating neuroprotection efficacy of the
compound against multiple toxic dose range. The fact that
compound (−)-12 was able to rescue these cells strongly
indicates selective intracellular complexation of Fe(II) by the
compound to reduce oxidative stress. In this regard, it is
important to point out that such complexation with iron should
take place only intracellularly as media containing test drug was
Figure 4. Dose-dependent effect of (−)-12, 1 and (−)-pramipexole on cell viability of PC12 cells from toxicity induced by Fe3+/8HQ complex.
(a) Dose-dependent effect of Fe3+/8HQ complex on cell viability. PC12 cells were treated with varying concentrations of Fe3+/8HQ complex for
30 min followed by incubation for 24 h. (b−d) PC12 cells were pretreated with different doses of (−)-12, 1, and (−)-pramipexole respectively for 1 h
followed by treatment with 20/40 μM Fe3+/8HQ complex for 30 min and finally incubated for 24 h. The values shown are the mean SD of at least
three independent experiments performed with 4−6 replicates. One-way ANOVA analysis F(8, 63) = 554.8, p < 0.0001 for (a), F(7, 56) = 24.30, p <
0.0001 for (b), F(7, 56) = 29.75, p < 0.0001 for (c), and F(7, 55) = 36.52, p < 0.0001 for (d). ANOVA was followed by Tukey’s multiple comparison
post hoc test (****p < 0.0001, **p < 0.01, and *p < 0.05 compared to Fe3+/8HQ (20/40 μM) only group and ####p < 0.0001 compared to control
group).
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As mentioned above, Fe(III)-8HQ complex induces
oxidative stress via lipid peroxidation mechanism.44,47 There-
fore, we wanted to observe whether pretreatment of cells with
(−)-12 could reduce lipid peroxidation thereby protecting cells
from such oxidative stress. As shown in Figure 5b, treatment
with Fe/8HQ (30/60 μM) alone induced significant increase in
lipid peroxidation compared to the control. However, pretreat-
ment with (−)-12 reduced lipid peroxidation significantly in a
dose-dependent manner compared to treatment with Fe/8HQ
(30/60 μM) alone. This result clearly indicates the treatment
effect of the drug on reduction of oxidative stress induced by
Fe-8HQ complex (30/60 μM).
Having shown neuroprotection potential in the cellular
disease model, our next goal was to find out whether the drug
would be able to cross the blood brain barrier to be efficacious
in vivo. Reserpine induces depletion of catecholamines in nerve
terminals resulting in a cataleptic condition in rats, which is a
well-established and highly utilized animal model for PD-related
studies.48−50 Recently, pramipexole was found to improve
motor activity by reversing akinesia in reserpine-induced rats.51
In our present study, significant reduction of locomotion of the
rats was observed 18 h after the administration of reserpine
(5 mg/kg), which indicated the development of akinesia in rats
(Figure 6). Compound (−)-12 (10 μmol/kg) was not only
Figure 5. Dose-dependent effect of (−)-12 on cell viability of PC12
cells from toxicity and on lipid peroxidation of PC12 cells induced by
Fe3+/8HQ (30/60 μM) complex. (a) PC12 cells were pretreated with
different doses of (−)-12 for 1 h followed by treatment with 30/60 μM
of Fe3+/8HQ complex for 30 min and finally incubated for 24 h.
The values shown are the mean SD of at least three independent
experiments performed with 4−6 replicates. (b) Effect of pretreatment
with different concentrations of (−)-12 on thiobarbituric acid reactive
substances (TBARS) production induced by Fe3+/8HQ (30/60 μM)
Figure 6. Effects of (−)-12 upon reserpine (5 mg/kg, s.c., 18 h
pretreatment) induced hypolocomotion in rats. Each point represents
the mean SEM for three rats. Horizontal activity was measured as
described in the experimental section. Representation of horizontal
locomotor activity is at discrete 30 min intervals after the
administration of (−)-12 (10 μmol/kg, s.c.) compared to control
rats, 18 h after reserpine treatment. Difference between treatment was
significant by student’s unpaired two-tailed t test analysis of treatment
group to control, ***p < 0.0005.
complex. The values shown are the mean
SD of at least three
independent experiments. One-way ANOVA analysis F(7, 56) = 302,
p < 0.0001 for (a), F(4,5) = 527, p < 0.0001 for (b). ANOVA was
followed by Tukey’s multiple comparison post hoc test (****p <
0.0001, **p < 0.01 and *p < 0.05 compared to Fe3+/8HQ (30/60 μM)
only group and ####p < 0.0001 compared to control group).
removed prior to addition of Fe(III)-8HQ complex which
was then removed after 30 min and replaced by only media
solution.
highly efficacious in reversing akinesia in rats, compared to
reserpine treatment alone, but also demonstrated significant
enhancement of locomotion for the entire duration of the study
of 6 h. The mechanism of the observed locomotor stimulation
in the reserpine model is likely to be mediated by postsynaptic
D2/D3 receptor activation by (−)-12. Thus, the result suggests
that the compound is a potent agonist, which crosses the blood-
brain barrier effectively and provides a sustained locomotor
stimulation effect.
In conclusion, the absence of a disease-modifying drug for
PD makes the development of multifunctional neuroprotective
agents an important therapeutic strategy. To address this unmet
medical need and the emerging role of free iron(II) in PD, we
have shown here the development of a unique dopamine
agonist with preferential Fe2+ chelation properties. The data
presented here also shows that the compound is neuro-
protective in an in vitro model of neuronal PC12 cells treated
Interestingly, our previous Fe3+ chelating 8HQ derivative 1
could not rescue the PC12 cells from toxicity of Fe(III)-8HQ
complex (Figure 4c), indicating greater neuroprotection efficacy
of Fe2+ chelating bipyridyl moiety in (−)-12. Furthermore,
since (−)-pramipexole, a well-known antiparkinsonian drug,
constituted part of our hybrid agonist (−)-12, we wanted to
evaluate its effect in rescuing PC12 cells from toxicity of
Fe(III)-8HQ complex. Our results demonstrate pramipexole to
be much less neuroprotective in this experimental model
compared to (−)-12 (Figure 4d), reflecting contribution of
bipyridyl moiety in the compound for the enhanced neuro-
protection. The data strongly suggest a neuroprotective effect
from (−)-12 against toxicity induced by Fe-8HQ complex in
PC12 cells, and further validates the importance of iron(II)
chelation strategy for neuroprotective treatment of PD.
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with the neurotoxin Fe(III)-8HQ complex presumably by
chelating intracellular iron(II) delivered by Fe(III)-8HQ
complex. Interestingly, under the same experimental condition
(−)-12 produced greater degree of neuroprotection compared
to 1 and (−)-pramipexole, reflecting superior neuroprotection
of (−)-12 due to complexation to Fe2+ which plays an impor-
tant role in PD neurodegeneration.36,52 Part of this neuro-
protection mechanism might also be explained by the effect of
the drug in reducing oxidative stress. In a reserpinized PD
animal model, the lead compound was highly efficacious in
reversing hypolocomotion with a long duration of action. This
report, therefore, underpins the notion that a multifunctional
drug like (−)-12 has the potential not only to ameliorate motor
dysfunction in PD patients, but also to modify disease
progression by protecting DA neurons from neurotoxic insults
in addition to restoring their function.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acschemneuro.6b00356.
1
Experimental procedures; H NMR, 13C NMR, ESI-MS,
and elemental analysis for the lead molecules; details of
in vitro and in vivo assay procedures; additional in vitro
data (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
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*Tel: 313-577-1064. Fax: 313-577-2033. E-mail: adutta@
wayne.edu.
ORCID
Aloke K. Dutta: 0000-0003-3347-0538
Author Contributions
A.K.D. was involved with all aspects of the studies including
design of compounds and biological (in vitro and in vivo)
experiments reported in the manuscript. B.D. was involved with
synthesis of the compounds. B.D. and L.X. carried out in vitro
cell culture and in vivo biological experiments. T.S., A.K.,
A.K.D., and B.D. carried out iron complexation studies.
M.E.A.R. and T.A. screened the compounds at dopamine D2
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Funding
This work is supported by National Institute of Neurological
Disorders and Stroke/National Institute of Health (NS047198,
A.K.D.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Dr. K. Neve, Oregon Health and Science
University, Portland, OR, for D2L and D3 expressing HEK cells.
We are also grateful to Dr. J. Shine, Garvan Institute for
Medical Research, Sydney, Australia, for D2L expressing CHO
cells. Initial contribution toward the synthesis of the
compounds by Drs. Shahid Islam and Soumava Santra is
acknowledged.
G
DOI: 10.1021/acschemneuro.6b00356
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
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