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. 2015 Aug 10;6(9):1025–1029. doi: 10.1021/acsmedchemlett.5b00281

Novel Fluorinated 8-Hydroxyquinoline Based Metal Ionophores for Exploring the Metal Hypothesis of Alzheimer’s Disease

Steven H Liang †,‡, Adam G Southon §, Benjamin H Fraser ∥, Anwen M Krause-Heuer ∥, Bo Zhang ∥, Timothy M Shoup †,‡, Rebecca Lewis †, Irene Volitakis §, Yifeng Han ⊥, Ivan Greguric ∥, Ashley I Bush §,*, Neil Vasdev †,‡,*
PMCID: PMC4569883  PMID: 26396692

Abstract

graphic file with name ml-2015-00281m_0009.jpg

Zinc, copper, and iron ions are involved in amyloid-beta (Aβ) deposition and stabilization in Alzheimer’s disease (AD). Consequently, metal binding agents that prevent metal-Aβ interaction and lead to the dissolution of Aβ deposits have become well sought therapeutic and diagnostic targets. However, direct intervention between diseases and metal abnormalities has been challenging and is partially attributed to the lack of a suitable agent to determine and modify metal concentration and distribution in vivo. In the search of metal ionophores, we have identified several promising chemical entities by strategic fluorination of 8-hydroxyquinoline drugs, clioquinol, and PBT2. Compounds 15–17 and 28–30 showed exceptional metal ionophore ability (6–40-fold increase of copper uptake and >2-fold increase of zinc uptake) and inhibition of zinc induced Aβ oligomerization (EC50s < ∼5 μM). These compounds are suitable for further development as drug candidates and/or positron emission tomography (PET) biomarkers if radiolabeled with 18F.

Keywords: 8-Hydroxyquinoline, metal ionophore, positron emission tomography, Alzheimer’s disease

Alzheimer’s disease (AD), a neurodegenerative disorder that affects approximately 44 million people worldwide, is the sixth leading cause of death with an estimated socioeconomic burden of more than $200 billion. There is no cure for the debilitating disease with only few symptom-alleviating treatments.1−4 Thus, understanding of disease etiology and development of therapeutics and biomarkers for AD is urgently needed.

AD is characterized by extracellular amyloid plaques containing Cu and Zn, which is accompanied by neuronal Cu deficiency and Zn dys-homeostasis.5−9 It is known that Zn and Cu ions are involved in the Aβ deposition and stabilization and that metal chelating agents can lead to the dissolution of Aβ deposits by preventing metal–Aβ interaction.10−13 Therefore, the metal hypothesis of AD has led to the search for diagnostic and therapeutic agents that are able to modulate or redistribute metal ions within the brain. A prototypical metal-chelating drug, 5-chloro-7-iodo-quinolin-8-ol (clioquinol; CQ (11); Chart 1), prevents Aβ toxicity. The metal ionophore activity of CQ (11) promotes cellular Zn and Cu uptake, initiating protective cell signaling events to degrade Aβ and prevent toxicity.6,14 In a pilot phase II clinical trial, CQ (11) was well tolerated and attenuated the rate of cognitive decline in AD patients; however, further development was halted due to a contaminant during the manufacturing process.15 A newer generation metal chelator, PBT2 (5,7-dichloro-2-((dimethylamino)methyl) 8-quinolinol; 18), has also shown benefits in patients with Huntington’s disease and patients with AD in phase II clinical trials.16,17

Chart 1. Representative Metal Chelators and Radiolabeled Derivatives for AD (A) and Design Strategy (B).

Chart 1

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To identify a suitable metal chelator for AD drug development, a radiopharmaceutical based on a metal chelator would be useful to determine metal concentration and distribution in the living brain by positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Development of such agents would also advance our understanding of AD etiology that are affected by dysregulation of metal functions and may prove useful in monitoring therapeutic response and disease progression for patients with AD. The first attempt to achieve this goal was carried out with iodine-123 labeled CQ and evaluated as an imaging marker in human subjects (Chart 1). Unfortunately, radio-deiodination in vivo and low brain uptake in human subjects precluded the use of this radiopharmaceutical for further development.18,19 Our laboratory reported the first analogous PET radiotracer labeled with fluorine-18 (18F, β+, t1/2 = 109.7 min) for this target, 2-[18F]fluoro-8-hydroxyquinoline ([18F]CABS13; Chart 1A), which demonstrated a significant temporal difference in brain uptake and retention among a transgenic mouse model of AD (APPswe/PSEN1dE9) compared with wild-type control.20,21 However, PET imaging studies in normal nonhuman primates revealed low brain uptake of the radiotracer and fast metabolism, which may be attributed to species differences.22 Notably, preliminary evaluations of other PET radiotracers to probe the metal hypothesis of AD were subsequently evaluated. Lim et al. recently reported two small molecules, namely, N-(pyridin-2-ylmethy)aniline (L2-a) and N1,N1-dimethyl-N4-(pyridin-2-ylmethyl)benzene-1,4-diamine (L2-b), based on a hybrid design of CQ (11) and stilbene derivatives (Chart 1A). These compounds modulated metal induced Aβ aggregation and toxicity in vitro. In particular, L2-b demonstrated the ability to dissolve Aβ aggregates from brain tissue homogenates from AD patients.23 Scott et al. radiolabeled compound L2-b with carbon-11 (11C, β+, t1/2 = 20.3 min) or 18F and performed a preliminary evaluation in healthy nonhuman primates,24 and Donnelly et al. combined a functionalized styrylpyridine group and copper-64 (64Cu, β+, t1/2 = 12.7 h) labeled thiosemicarbazone to detect Aβ in vtiro in post-mortem brain tissue of AD patients.25

The goal of present work is to synthesize a series of fluorinated 8-hydroxyquinolines as metal selective chelators for therapeutic applications and as potential PET imaging agents. Our design strategy is based on lead compounds 8-hydroxyquinoline (8HQ; 1), CQ (11), and PBT2 (18). We expect to increase binding affinity, metal selectivity, and in vivo stability by strategically placing functional groups, including fluorine atom around hydroxyquinoline core, as well as design a better chelating interaction with metals, i.e., Zn and Cu, by increasing binding ability (Chart 1B). Two in vitro methods were used to evaluate these new compounds. Ionophore activity was assessed in cultured SH-SY5Y cells, a well-established neuronal model system. Cells were treated with each compound, and inductively coupled plasma mass spectrometry (ICP-MS) was used to identify compounds that promoted cellular uptake of Zn and Cu (Figures 1–3). While the compounds were not expected to be Fe chelators, cellular Fe uptake was also measured. Aggregation of Aβ oligomers was assessed fluorometrically with 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate (bis-ANS). The effective concentration that reversed Aβ aggregation by 50% (EC50) was determined with nonlinear regression analysis (Table 1 and Supporting Information).

Figure 1.

Figure 1

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Ionophore assay of 8HQ derivatives. Dashed line indicates control level. SH-SY5Y cells were treated with each compound (20 μM) for 6 h, and cellular metal levels were measured with ICP-MS. Dashed line indicates control level.

Figure 3.

Figure 3

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Ionophore activity of PBT2 derivatives. SH-SY5Y cells were treated with each compound (20 μM) for 6 h, and cellular metal levels were measured by ICP-MS. Dashed line indicates control level.

Table 1. Reversal of Aggregation of Soluble Aβ Oligomers by Fluorinated Hydroxyquinoline Derivativesa.

Cpd. No. EC50 ± SD (μM) Cpd. No. EC50 ± SD (μM)
CABS13 (5) >120 15 5.2 ± 0.5
6 43.5 ± 4.4 16 3.7 ± 0.4
7 21.5 ± 15.9 17 2.5 ± 2.8
8 37.5 ± 10.1 PBT2 (18) 2.0 ± 0.3
9 14.0 ± 3.2 23 2.4 ± 0.4
10 15.4 ± 3.4 24 2.0 ± 0.4
CQ (11) 1.8 ± 0.4 28 2.3 ± 0.4
12 4.9 ± 1.8 29 4.5 ± 1.3
13 3.5 ± 0.6 30 2.8 ± 0.1
14 2.3 ± 0.4    
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a

Aggregation of Zn-induced Aβ oligomers was assessed fluorometrically with bis-ANS. CQ and PBT2 were used as positive controls. EC50 ± SD were determined with nonlinear regression analysis (Prism 6, Graphpad). All compounds had an EC50 significantly lower than CABS13, P < 0.01.

Herein we describe an array of candidate compounds that showed superior binding affinity, metal selectivity and Cu and Zn ionophore activity over the therapeutic drugs CQ (11) and PBT2 (18). These promising compounds provide a candidate pool for the development of drug candidates and/or PET ligands. As shown in Chart 2, efforts were first focused on fluorinated 8-hydroxyquinoline derivatives to improve the ionophore ability. We discovered that the binding pocket was sensitive to different heteroaromatic rings (compounds 2 and 3). We then systematically evaluated fluorine substituents around the hydroxyquinoline because fluorine contribution to the electron density of the heterocyclic ring is a function of its position on 8HQ (1). CABS13 (compound 5) and compound 6 were inferior to 8HQ (1) in terms of Cu uptake, while compounds 7–9 showed equal or superior Cu uptake (Figure 1). Neuronal Zn and Fe uptake were largely unaffected. Modification of CQ was carried out in attempt to discover lead candidate radiotracers, which can reveal higher brain uptake than [123I]CQ.19 Therefore, we replaced labile iodine on CQ with other halides, such as fluoride, chloride, or bromide, at various positions to improve the stability of the molecule due to higher C–X (X = F, Cl or Br) bond energies (Chart 3). Among six analogues screened, positional isomers 15–17 are of particular interest because of improved Cu and Zn ionophore activity (Figure 2).

Chart 2. Structures of 8-Hydroxyquinolines and Related Compounds.

Chart 2

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Chart 3. Structures of 8HQ Derivatives.

Chart 3

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Figure 2.

Figure 2

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Ionophore activity of CQ derivatives. SH-SY5Y cells treated were treated with each compound (20 μM) for 6 h, and cellular metal levels were measured with ICP-MS. Dashed line indicates control level.

Since PBT2 (18) is characterized as a monoprotic tridentate ligand, we proposed several new molecular entities by changing the nature of the coordination site or denticity to achieve better metal binding affinity and selectivity toward Cu and Zn ions. Specifically, substitution with chelating properties on the 2-position of the pyridine ring would expect to increase the interaction with metals, i.e., Zn or Cu, in brain. The change from bidentate to tridentate, even tetradentate, could ensure enhanced ionophore ability greater than that of CQ. Two parallel approaches, namely, fluoroalkyl (compounds 23 and 24) and fluorotriazole (compounds 28–30) derivatives, were prepared. As indicated in Scheme 1, compound 20 was first protected with a Boc group, followed by SeO2 oxidation, to yield aldehyde 21. After NaBH4 reduction and subsequent alkylation with the corresponding fluoroalkyl tosylate, compounds 23 and 24 with different aliphatic fluorine atom were achieved in 16–18% yield. Three triazole derivatives were accessed by a similar manner using Huisgen cycloaddition (click chemistry) to give compounds 28–30 in 39–49% yields. To our delight, fluorinated compound 24 exhibited similar ionophore behavior compared to PBT2 (18). Compounds 28–30 showed exceptional Cu uptake (10-fold increase for compound 28, 16-fold increase for compound 29, and 8-fold increase for compound 30) greater than that of PBT2 (18). For Zn neuronal uptake, compounds 28–30 showed comparable results to those of PBT2 (18) (Figure 3). While none of the fluorinated compounds promoted Fe uptake in these assays, Fe ionophore activity cannot be excluded. Importantly, the Cu and Zn ionophore activity of several compounds was comparable to the clinically relevant 8-hydroxyquinolines CQ (11) and PBT2 (18).

Scheme 1. Synthesis of PBT2 derivatives.

Scheme 1

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Conditions: (a)Boc2O, DMAP, THF, 90%; (b) SeO2, 1,4-dioxane; (c) NaBH4, then TFA, 80% from step b; (d) NaH, DMF, fluoroalkyl tosylate, 16% (n = 1), 18% (n = 3); (e) CH3NH2, THF, 93% from step b; (f) NaBH4, MeOH, 58%; (g) 3-bromopropyne, DIPEA, DMF, 33%; (h) fluoroalkyl azide, CuSO4, Na ascorbate, THF/H2O, 45% (n = 1), 49% (n = 2), 39% (n = 3).

Fluorinated compounds were also evaluated for their ability to reverse the aggregation of soluble Aβ oligomers in vitro (Table 1 and Supporting Information). From our previous PET imaging studies, [18F]CABS13 detects Aβ in vivo,20,25 but the present study showed that this compound does not affect Aβ aggregation in vitro and metal uptake. However, fluorinated 8-hydroxyquinolines (Chart 2, compounds 6–10) improved anti Aβ aggregation activities compared to CABS13 (compound 5). Three fluorinated CQ analogues (Chart 3, compounds 12–14) and another set of fluoro analogues (compounds 15–17) have EC50 values similar to CQ in the range of 2.3–5.2 μM. PBT2 derivatives 23, 24, and 28–30 (EC50 = 2.0–4.5 μM) are also excellent candidates. Compounds 28–30 may be regarded as an improvement on PBT2, as they display improved Cu ionophore activity while retaining similar Aβ disaggregation activities.

In summary, we have synthesized an array of fluorinated hydroxyquinolines based on the clinical CQ (11) and PBT2 (18) scaffolds. Several equipotent lead compounds, 15–17 and 28–30, identified from the ionophore and Aβ reversal assays are worthy of further evaluation as potential therapeutics and/or PET ligand development to probe the metal hypothesis of AD.

Glossary

ABBREVIATIONS

PET

positron emission tomography

AD

Alzheimer’s disease

Aβ

amyloid-β peptide

CQ

clioquinol

PBT2

5,7-dichloro-2-((dimethylamino)methyl) 8-quinolinol

Boc2O

di-tert-butyl dicarbonate

DMAP

4-dimethylaminopyridine

THF

tetrahydrofuran

TFA

trifluoroacetic acid

DMF

dimethylformamide

DIPEA

N,N-diisopropylethylamine

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.5b00281.

  • Synthetic details and characterization of all new compounds and assay methods (PDF)

Author Contributions

# These authors contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest: Dr. Bush is a shareholder in Prana Biotechnology Ltd., Cogstate Ltd., Mesoblast Ltd., Eucalyptus Ltd. and Cogstate Ltd., and a consultant for Collaborative Medicinal Development Pty Ltd.

Supplementary Material

ml5b00281_si_001.pdf (1.2MB, pdf)

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Associated Data

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Supplementary Materials

ml5b00281_si_001.pdf (1.2MB, pdf)

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