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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 L. Stemmler, Maarten E.A. Reith, and Aloke K. Dutta ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00356 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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ACS Chemical Neuroscience

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, MI 48202

‡

Department of Psychiatry, New York University, New York, NY 10016

*Corresponding Author: Aloke K. Dutta, Ph.D. Department of Pharmaceutical Sciences Eugene Applebaum College of Pharmacy & Health Sciences Wayne State University Detroit, MI 48202 Tel: 1-313-577-1064, Fax: 1-313-577-2033, e-mail: [email protected]

Keywords: Parkinson’s disease, D2/D3 agonist, iron chelation, neuroprotection, multifunctional drug

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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 multitargetdirected 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 almost 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.

Introduction Parkinson’s disease (PD) is a major neurodegenerative disorder 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 studies10. 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 (αSyn) protein to form toxic oligomers.12, 13 Therefore, the critical cross talk between iron accumulation and neurodegeneration suggests that iron chelation might be an attractive therapeutic strategy to be used against the progression of PD.14 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, 2 ACS Paragon Plus Environment

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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 FDA so far for the treatment 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 with slowing or halting the neurodegeneration process by our multifunctional drug development approach.21-26 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-1,2,3,6tetrahydropyridine)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 development 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 head group is attached to a bipyridyl moiety, a known iron chelating agent having preferential affinity for Fe2+.32-35 The rationale for employing the bipyridyl fragment 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 generate reactive oxygen species.37 Thus, a Fe2+ preferring chelator should provide greater beneficial effect against neurodegeneration. 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 ironinduced 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. 3 ACS Paragon Plus Environment


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Results and Discussion Our attempt to find a multifunctional compound was based on the intracellular 2+

Fe

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 (2bromoethoxy)-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 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 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 compounds were characterized by 1H NMR,

13

C NMR, HRMS and

elemental analysis (refer to Supporting Information for detail). 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 [3H]spiroperidol binding to rat dopamine D2 and D3 receptors expressed in human embryonic kidney-293 (HEK293) 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 Supporting Information). A similar profile was also observed for compound (-)-13 (Ki, 4 ACS Paragon Plus Environment

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D2 = 227 nM, D3 = 8.90 nM, D2/D3 = 25). On the other hand, compound (-)-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 towards D2 than D3 receptors compared to the 2aminothiazole 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 monitoring stimulation of [35S]GTPγS (guanosine 5'-O-[gammathio]triphosphate) binding in comparison to stimulation by the full agonist DA.21 As shown in Table 2 and Figure S4B in 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. 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 conditions 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 2C for Fe(III) and Fe(II), 5 ACS Paragon Plus Environment


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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 Equation 1 within Dynafit42 for a 1:3 Fe(II) vs. ligand stoichiometric profile. In equation 1, L is the compound (-)-12 and Kd1 is the dissociation constant for the Fe(II) complexation reaction. Simulations of the representative data in Figure 2, inset c resulted in a calculated Kd of 1.60 (± 0.33) x 10-13 M for Fe(II) binding to compound (-)-12. Kd1

Fe(II)-LLL

Eqn 1

Fe(II) + L + L + L

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 profile were fit using Equation 2 with a 1:1 Fe(III):ligand stoichiometry 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) x 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 publications.

with the literature

33-35

Kd2 Fe(III)-L

Fe(III) + L

Eqn 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 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) 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 production and lipid peroxidation;44,

46, 47

however, such a phenomenon 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.


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Here we show that treatment of PC12 cells with increasing concentrations of (-)12 alone (0.1–10 µM) showed no significant cell loss compared to untreated control (Figure 3c), indicating the non-toxic profile of the compound at the doses tested. Moreover, iron or 8HQ (8-hydroxyquinoline) alone only conferred appreciable toxicity at a higher concentration range (Figure 3a and 3b, 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 experiments (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 pre-treated 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 removed prior to addition of Fe(III)-8HQ complex which was then removed after 30 min and replaced by only media solution. 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



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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 neuroprotection. 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. As mentioned above, Fe(III)-8HQ complex induces oxidative stress via lipid peroxidation mechanism.44,

47

Therefore, we wanted to observe whether pre-

treatment 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, pre-treatment with (-)-12 reduced lipid peroxidation significantly in a dosedependent 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 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) 8 ACS Paragon Plus Environment

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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 neuroprotective in an in vitro model of neuronal PC12 cells treated 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 important role in PD neurodegeneration.36,

52

Part of this

neuroprotection 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.

ASSOCIATED CONTENT Supporting Information Experimental procedures; 1HNMR,

13

CNMR, ESI-MS and elemental analysis for the

lead molecules; details of in vitro and in vivo assay procedures; additional in vitro data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: (+1) 313-577-1064. Fax: (+1) 313-577-2033.

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



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D2 and D3 receptors. B.D. and A.K.D. participated in writing the manuscript. M.E.A.R. provided some editing comments of the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgements This work is supported by National Institute of Neurological Disorders and Stroke/ National Institute of Health (NS047198, AKD). 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 towards the synthesis of the compounds by Drs. Shahid Islam and Soumava Santra is acknowledged.

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Figure Legends Figure 1. Structures of iron chelators. 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.



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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 3a, F (8, 63) = 82.98, p < 0.0001 for 3b and F (8, 62) = 9.89, p < 0.0001 for 3c. ANOVA was followed by Tukey’s multiple comparison post hoc tests (#p < 0.05, ##p < 0.01, ###p < 0.001 and ####p

A Novel Iron(II) Preferring Dopamine Agonist ... - ACS Publications

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