Preparation and Characterization of Manganese (II) Complexes with Potential

Therapeutic Application against α-Synuclein Aggregation

Kate Byrne

BSc Medicinal Chemistry & Pharmaceutical Science

Year 3 Project

Overview

• Aim of the Project

• Parkinson’s Disease and Etiology

• α-Synuclein and Lewy Bodies

• Project Rationale and Objectives

• Synthesis of the compounds

• Characterisation of the compounds

• Conclusion

Aim

The aim of this project was to synthesize, purify and characterise three manganese(II) complexes as part of a structure-activity relationship (SAR) study of a potential

therapeutic system for Parkinson’s disease

Parkinson’s Disease Parkinson’s disease, (PD) is a progressive neurological

disease which is a result of the loss of dopamine producing brain cells

Parkinson’s disease is named after Dr. James Parkinson who first reported symptoms of the disease in 1817 when

it was referred to as “Paralysis Agitans”

Etiology Idiopathic

Figure 1: Dr. James Parkinson

Statistics

• Parkinson’s disease affects 6.3 million people worldwide.

• Over 9,000 of those affected by the disease are Irish.

• The disease typically develops at the age of 65 however people can develop “early-onset” parkinson’s before reaching 50 years.

• Current life expectancy: 81 years

• Projected Life expectancy 2030: 90+ years

α-Synuclein α-Synuclein is a protein that is abundant in the human brain.

In the early stages of Parkinson’s disease misfolded α-synuclein

proteins are converted to pathological oligomers and higher

order aggregates that form fibrils leading to the formation of

Lewy Bodies.

Figure 2: α-Synuclein aggregation stages

Emil Paleek, et al., Changes in interfacial properties of α-synuclein preceding its aggregation, Analyst, 2008, 133, 76.

Lewy bodies are abnormal α-Synuclein protein aggregates that develop in nerve cells in regions of the brain that are

involved in motor control.

The presence of the Lewy Body protein aggregates in the

brain is a pathological hallmark of Parkinson’s Disease.1

Inhibiting α-Synuclein aggregation offers a therapeutic target for Parkinson’s Disease and evidence in the literature

suggests a role for inorganic medicinal chemistry2

1. He-Jin Lee, et al., Extracellular α-synuclein a novel and crucial factor in Lewy body diseases, Nature Reviews Neurology, 2014, 10, 92-98. 2. D.J. Haynes, S. Lim, P.S. Donnelly, Metal complexes designed to bind to amyloid-β for diagnosis and treatment of Alzheimer’s disease, Chem. Soc. Rev., 2014, 43, 6701-6715.

Lewy bodies and Parkinson’s Disease

Lead Compound

The complex salt, MD1 has been shown to be an excellent lead compound for the prevention of α-Synuclein aggregation.

MD1:

[Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2]4H2O

Where; odaH2 = octanedioic acid; phen = 1,10-phenanthroline

Figure 4: Structures of MD1 and Octanedioic Acid

• Comprised of bi-nuclear Cationic and Anionic components (Complex salt).1

• Bridging and terminal octanedioate ligands, deprotonated octanedioic acid

• Highly water soluble.

• Exhibits antioxidant capability.2

• Reduces α-Synuclein aggregation in fungal (saccharomyces cerevisiae) and mammalian (HEK293) cellular models.2

1. M. McCann, M. Devereux, et al. Synthesis and structure of the Mn2 (II,II) complex salt [Mn2(oda)(phen)4(H2O)2] [Mn2(oda)2(phen)4] (odaH2 = octanedioic acid): a catalyst for H2O2 disproportionation., J. Chem. Soc., Chem. Commun., 1994, 2643. 2. T. Ribeiro, M. McCann, M. Devereux, M. Pereira, et al., Evaluation of antioxidant activity of a Mn2+complex salt and its potential therapeutic use against alpha—synuclein aggregation. Manuscript in preparation. (Target Journal: Nature Communications)

Research question

What are the structural aspects of MD1 that are important for its

α-Synuclein aggregation inhibitory properties?

Objective of the Structure-Activity Relationship Study

To investigate analogues of MD1 to determine how variations in structure influence the α-Synuclein aggregation inhibitory potential of this class of

manganese(II) complex.

Note: The analogues were varied in terms of the number of Manganese centres in the complex as well as the charge

on the complex

Key Objectives

To synthesise and characterise three known analogues of MD1 with variations in structure achieved by replacing the octanedioate ligands with aliphatic

dicarboxylic acid ligands of varying chain length

[Mn(hxda)(phen)2(H2O)].7H2O 1

[Mn(pda)(phen)]2

[Mn2(bda)2(phen)2(H2O)].2H2O3

Hexanedioic acid (hxdaH2)

Pentanedioic acid (pdaH2)

Butanedioic acid (bdaH2)

1. M. McCann, M. Devereux, et al., Manganese(II) complexes of hexanedioic and heptanedioic acids: X-ray crystal structure of [Mn(hxda)(phen)2(H2O)].7H2O and [Mn(phen)2(H2O)][Mn(hpda)(phen)2(H2O)](hpda) .12.5H2O, Polyhedron, 1997, 16, 2741.

2. Martin Curran, PhD Thesis, DIT/NUIM 1996.

3. M. McCann, M. Devereux, et al., Synthesis, X-ray crystal structure and catalytic activities of manganese(II) butanedioic acid complexes [Mn(bda)(phen)2(H2O)4].2H2O and {[Mn(bda)(bipy2(H2O)2].H2O }n, Polyhedron, 1997, 16, 2547.

Octanedioic acid (odaH2)

Structure of the Analogue Complexes

[Mn(hxda)(phen)2(H2O)].7H2O : Mononuclear (1 manganese centre), 2 phens per manganese, neutral charge

[Mn(pda)(phen)]: Polymeric, 1 phen per manganese, neutral

charge.

[Mn2(bda)2(phen)2(H2O)].2H2O: Binuclear (2 manganese centres), 1 phen per

manganese, neutral charge

Synthetic Scheme Stage One: Generation of the manganese(II) dicarboxylate

precursors

Where; R = -(CH2)4 - or -(CH2)3 -or -(CH2)2 -

Synthetic Scheme Stage Two: Reaction of the precursor complexes with 1,10-

phenanthroline to produce the analogues of MD1

{Mn(OOC-(CH2)n-COO)}x + 4 phen → [Mn(hxda)(phen)2(H2O)].7H2O (where n = 4) [Mn(pda)(phen)] (where n = 3) [Mn2(bda)2(phen)2(H2O)].2H2O (where n = 2)

EtOH

Reflux

Characterisation

The three precursor complexes and the three analogue complexes were characterised using the following

techniques:

• Infra-Red (IR) Spectroscopy

• Magnetic Susceptibility Analysis

• Inductively-Coupled Mass Spectrometry (ICP-MS)

Infrared Spectroscopy An IR spectrum was recorded of:

• The Ligand

• The Precursor Complex

• The Final Complex

Each of the spectra was then overlayed

Hexanedioic Acid

[Mn(hxda)].H2O

[Mn(hxda)(phen)2(H2O)].7H2O

Hexanedioic Acid Spectra Overlay

Infrared Spectroscopy

Precursor Complex and Analogue Complex Similarities

Characteristic Carbonyl Peak

• Asymmetric Stretch 1590-1547cm-1

• Symmetric Stretch 1400-1420cm-1

Determination of [Mn] Using ICP

y = 66202x + 5669.9

R² = 0.9999

0

100000

200000

300000

400000

500000

600000

-1 0 1 2 3 4 5 6 7 8 9

Intensity

Concentration (ppm)

ICP Standards

Mn2+ Analysis Name of Complex Theoretical

Concentration

(ppm)

Experimental

Concentration

(ppm)

[Mn(bda)].2H2O 5.3 4.647

[Mn(pda)].H2O 5.36 4.001

[Mn(hxda)].H2O 5 3.468

Drug 1,

[Mn2(bda)2(phen)2(H2O)].2

H2O

5.4 9.5

4.75 x 2

Drug 2, [Mn(pda)(phen)] 5.45 4.325

Drug 3,

[Mn(hxda)(phen)2(H2O)].7

H2O

4.68 1.468

Magnetic Susceptibility Balance

µ𝑠 .𝑜. = 𝑛(𝑛 + 2)

Where;

N = number of unpaired electrons, n=5

µ𝑠 .𝑜. = 5(5 + 2) = 35 = 5.92

µs.o. = 5.92B.M.

Gram Magnetic Susceptibility Calculation

Χg = c ∗ l ∗ (R − R0)

109 ∗ m

C= calibration constant, 1.05

L= sample length, cm

R= reading taken of sample

R0 = reading taken with no sample

M= sample mass (g)

𝛸𝑀 = 𝛸𝑔 ∗ 𝑀

Where;

𝛸𝑀 = Molar Magetic Susceptibility

𝛸𝑔= Gram Magnetic Susceptibility (cm3 g-1)

M= Molar Mass (g mol-1)

Molar Magnetic Susceptibility

Effective Magnetic Moment

µEFF = 2.828 𝑇𝑋𝑀

µEFF = effective magnetic moment, B.M.

T= Temperature (K), 292K

XM = Molar magnetic Susceptibility, cm3 mol-1

Name of Substance Theoretical Value

B.M.

Experimental

Value B.M.

[Mn(bda)].2H2O 5.92 5.87

[Mn(pda)].H2O 5.92 6.16

[Mn(hxda)].H2O 5.92 3.09

Drug 1,

[Mn2(bda)2(phen)2(H2O)].

2H2O

5.92 8.91

4.455 x 2

Drug 2, [Mn(pda)(phen)] 5.92 6.36

Drug 3,

[Mn(hxda)(phen)2(H2O)].

7H2O

5.92 8.62

Conclusion

1. The complex salt [Mn2(oda)(phen)4(H2O)2] [Mn2(oda)(phen)4(oda)2]4H2O (MD1) is an excellent lead candidate for the prevention of α-Synuclein aggregation.

2. Three known analogues of MD1 {[Mn(hxda)(phen)2(H2O)].7H2O; [Mn(pda)(phen)]; and [Mn2(bda)2(phen)2(H2O)].2H2O along with their precursor manganese(II) dicarboxylate complexes have been synthesised using methods previously published

3. All six complexes have been characterised using a range of analytical techniques.

4. Further work is required to purify the complexes in preparation for their use in a structure-activity relationship study.

Acknowledgements

I would like to thank my supervisors Professor Michael Devereux and Dr. Patricia Ennis for their help and support

when carrying out this project.

I would also like to thank the School of Chemical & Pharmaceutical Sciences for allowing me to use their

equipment.

Thank You