2-Phenylbenzothiazolyl iridium complexes as inhibitors and probes of amyloid β aggregation

Abstract

The aggregation of amyloid β (Aβ) peptides is a significant hallmark of Alzheimer's disease (AD), and the detection of Aβ aggregates and the inhibition of their formation are important for the diagnosis and treatment of AD, respectively. Herein, we report a series of benzothiazole-based Ir(III) complexes HN-1 to HN-8 that exhibit appreciable inhibition of Aβ aggregation in vitro and in living cells. These Ir(III) complexes can induce a significant fluorescence increase when binding to Aβ fibrils and Aβ oligomers, while their measured log D values suggest these compounds could have enhanced blood–brain barrier (BBB) permeability. In vivo studies show that HN-1, HN-2, HN-3, and HN-8 successfully penetrate the BBB and stain the amyloid plaques in AD mouse brains after a 10-day treatment, suggesting that these Ir(III) complexes could act as lead compounds for AD therapeutic and diagnostic agent development.

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Introduction

Alzheimer's disease (AD) is the most common neurodegenerative disease and the 7^th leading cause of death in the US.¹ One strategy for the treatment of AD is the development of inhibitors that prevent the aggregation of monomeric Aβ peptides into neurotoxic Aβ species.^2–12 Transition metal complexes have emerged as a viable alternative to organic compounds with distinct biological properties and have been wildly utilized for the treatment of cancer.^13,14 Recently, transition metal complexes have been developed as chemical reagents capable of altering Aβ aggregation.^15–38 Among many of these transition metal complexes, cyclometalated Ir(III) complexes would be promising candidates as their photophysical and photochemical properties are unique and desirable. In general, these complexes exhibit high luminescence quantum yields and long lifetimes due to the efficient spin–orbit coupling induced by the heavy Ir(III) center. Also, they have high thermal and chemical stability, as Ir(III) is a substitutionally inert transition metal ion.^39,40 Moreover, tuning the emission color and excited-state dynamics is easily achieved through ligand structure modification, by either increasing conjugation, introducing heteroatoms, and/or various substituents. The preparation of Ir(III) complexes is highly modular when compared to the multi-step and linear synthesis of organic molecules.^41,42 This synthetic versatility could be exploited to enhance the interaction of Ir(III) complexes with Aβ species, which could be employed for controlling the Aβ aggregation pathways.^29,43,44

In this work, eight Ir(III) complexes that contain Aβ-binding heterocycle ligands were designed and synthesized. These complexes with two or three Aβ binding groups are stable and exhibit optimal log D values and appreciable affinity for Aβ fibrils and Aβ oligomers. Importantly, in vivo studies have shown that four of the Ir(III) complexes specifically label the amyloid species in the brains of transgenic AD mice.

Results and discussion

Compounds design, synthesis and characterization of Ir(III) complexes

Complexes HN-1 to HN-3, with 2-phenyl-1,3-benzothiazole as the ‘C^N’ ligand, were first designed and synthesized (Fig. 1, see ESI† for experimental details). HN-1 is a cationic Ir(III) complex with two labile acetonitrile (MeCN) ancillary ligands, which allow for potential coordination to the Aβ amino acid residues. Then, HN-2 was designed as a neutral tris-cyclometalated Ir(III) complex with increased lipophilicity and the potential to cross the blood–brain barrier (BBB). HN-3 was also a neutral complex with an acetylacetonate (acac) ancillary ligand, which could be further modified. Complexes HN-4 to HN-6 were constructed by extension of the 2-phenyl-1,3-benzothiazole C^N ligand with a pendant aromatic fragment. We proposed that the pendant moiety could rotate freely in solution, yet its free rotation would be hindered when the corresponding Ir(III) complexes would interact with the Aβ fibrils, and thus a fluorescence “turn-on” effect would be observed. Complex HN-8 was designed by replacing the acac ligand with curcumin, a natural product which has been reported to have the ability to alleviate Aβ toxicity (Scheme 1).^45–47 Importantly, replacing the acac ligand with other anionic chelating ligands is practical, since attaching the acac-related ligand is the last step in the synthesis of these inert Ir(III) complexes.

Fig. 1 Rational design strategies of Ir(III) complexes.

Scheme 1 Synthetic routes to Ir(III) complexes. (a) Complexes HN-1–7; (b) complex HN-8.

The molecular structure of HN-1 was determined by single crystal X-ray diffraction (Fig. 2). The Ir atom adopts a distorted octahedral coordination geometry, with two C^N cyclometalated ligands and two acetonitrile ligands bound to the Ir center. The C^N nitrogen atoms are in a trans orientation relative to each other. The Ir–C and Ir–N bond lengths of HN-1 are similar to those of the previously reported Ir(III) complexes with the 2-phenyl-1,3-benzothiazole C^N ligand, ranging between 2.007 and 2.143 Å.^42,48,49

Fig. 2 X-ray crystal structure of HN-1. Thermal ellipsoids are drawn at the 50% probability with solvent molecules, counter ion and hydrogen atoms are eliminated for clarity. Selected bond distances (Å): Ir1–C1, 2.007(3); Ir1–C14, 2.008(3); Ir1–N1, 2.061(3); Ir1–N2, 2.064(3); Ir1–N3, 2.128(3); Ir1–N4, 2.143(3).

Photophysical and biochemical properties of Ir(III) complexes

The UV-vis absorption spectra of HN-1 to HN-8 were obtained in PBS with 0.5% DMSO (Fig. S1†).⁵⁰ Complexes HN-1, HN-2, HN-3, and HN-8 display intense absorption bands around 320 nm that can be assigned to spin-allowed ligand-centered π–π* transitions (¹LC). Due to the varied backbone C^N ligand for complexes HN-4 to HN-7, the spin-allowed ligand-centered π–π* transitions (¹LC) occur around 370, 350, 355, and 335 nm, respectively. Less intense charge transfer (^1,3CT) absorption bands around 500 nm can be observed in the spectra for all eight complexes.

These complexes exhibit emission in the 520–700 nm region in PBS (Fig. 3a and Fig. S2†), where the Stokes shift is significantly large due to the phosphorescent properties of Ir(III) complexes.

Fig. 3 Fluorescence spectra of Ir(III) complexes with or without Aβ₄₂ fibrils (10 μM) or Aβ₄₂ oligomers (10 μM). (a) HN-1, (b) HN-2, (c) HN-4, and (d) HN-8.

As shown in Fig. 3a (black line), HN-1 showed an excellent turn-on response toward the Aβ₄₂ fibrils, displaying a nearly 200-fold fluorescence intensity increase (λ_em = 550 nm) for Aβ₄₂ fibrils and with a K_i value of 3.6 ± 0.3 μM (Fig. S7†).⁵⁰ This is likely due to the MeCN ligands in the Ir(III) complex being replaced by amino acid residues of the Aβ aggregates and therefore the Ir(III) complex coordinates to the peptides, as proposed previously.^51,52

In recent years, mounting evidence indicates that soluble Aβ oligomers are highly toxic for neurons.^53,54 Therefore, Ir(III) complexes developed in this study were also tested to see whether they could interact with soluble Aβ₄₂ oligomers. The Aβ₄₂ oligomers were prepared according to the procedure reported by Klein et al., and the morphology of obtained species was confirmed by transmission electron microscopy (TEM, Fig. S6†).⁵⁰ Surprisingly, HN-1 also showed a nearly 150-fold fluorescence intensity increase with soluble Aβ₄₂ oligomers (Fig. 3a, red line). Complexes HN-2 (Fig. 3b) and HN-3 did not exhibit a significant fluorescence increase when they bind to Aβ aggregates, which is also consistent with our previous assumption that the molecular structures of these complexes are too rigid. However, the K_i values of complexes HN-2 and HN-3 were similar to that of HN-1 at 6.0 ± 1.9 and 7.7 ± 1.0 μM, respectively (Fig. S7†), indicating they bind to Aβ fibrils with similar affinity due to interactions between the Aβ species and the 2-phenyl-benzothiazole ligand framework. As expected, the extended conjugated complexes HN-4 and HN-8 showed only slight turn-on effects when they bind to Aβ fibrils, with a 15-fold fluorescence increase for HN-4 (Fig. 3c), and a 20-fold increase for HN-8 (Fig. 3d), respectively.

Inhibition of amyloid β aggregation

The ability of the Ir(III) complexes to inhibit Aβ aggregation was probed via thioflavin T (ThT) fluorescence assays. ThT is a well-established fluorophore for the kinetic measurement of Aβ aggregation, and it is known that when it binds to amyloid fibrils, a pronounced enhancement of fluorescence can be detected at λ_max = 485 nm upon excitation at 435 nm. The aggregation of Aβ follows a sigmoidal curve, composed of three phases: a lag phase, an elongation phase, and a plateau phase (Fig. 4, black line). Compared to the control group, Ir(III) complexes HN-1 to HN-7 exhibit the ability to modulate Aβ aggregation to different extents. Excitingly, HN-1 completely quenched the Aβ aggregation (Fig. 4, red line, inhibition percentage: 100%), likely due to the replacement of acetonitrile ligands of HN-1 with histidine residues located at the N-terminus region of Aβ peptide, which play critical role in Aβ aggregation.⁵⁵ The remaining Ir(III) complexes HN-2 to HN-7 extended the lag phase and delayed the beginning of the elongation phase. By comparison, HN-8 exhibited a different behavior, likely due to the known decomposition of curcumin derivatives under physiological conditions (Fig. S10†). Importantly, we note that neither the excitation nor emission wavelengths of ThT overlap with those of the Ir(III) complexes.

Fig. 4 Time-resolved measurements of the aggregation of Aβ₄₂ through examination of ThT emission at 485 nm (λ_ex = 435 nm) for free Aβ₄₂ and Aβ₄₂ incubated with the ligands HN-1 to HN-7. Conditions: [Aβ₄₂] = 10 μM; [ThT] = 10 μM; [compound] = 10 μM.

5xFAD mouse brain section imaging

To examine whether the Ir(III) complexes could specifically interact with native Aβ species, brain section staining was performed. All brain sections were sliced from transgenic 5xFAD mice, which can overexpress human APP and PSEN1 transgenes. All of the eight Ir(III) complexes can specifically label Aβ plaques, showing excellent colocalization with staining of CF594 conjugated HJ3.4 antibody (CF594-HJ3.4) that binds to a wide range of Aβ species (Fig. 5).⁵⁶

Fig. 5 Fluorescence microscopy images of 5xFAD mice brain sections incubated with Ir(III) complexes. The fluorescence signals from Ir(III) complexes and CF594-HJ3.4 antibody were monitored under blue and red channels, respectively. Scale bar: 125 μm. R = Pearson's correlation coefficient.

log D measurements of Ir(III) complexes

The partition coefficients log D values were also examined to evaluate the BBB permeability of the Ir(III) complexes. For complexes HN-2 to HN-5, HN-7, and HN-8, log D values in the range of 0.9–2.5 were obtained (Table 1), suggesting these compounds are promising candidates for further in vivo studies. In the case of the cationic complex HN-1, as expected, a lower log D was obtained. HN-6 also showed a low log D value, presumably because of hydrogen bond formation through the methoxy group present on the ligand.

Table 1 Partition coefficients (log D) of Ir(III) complexes in octanol/PBS

Iridium complex	log D
HN-1	0.650 ± 0.110
HN-2	1.220 ± 0.055
HN-3	1.313 ± 0.067
HN-4	1.013 ± 0.084
HN-5	1.113 ± 0.029
HN-6	0.571 ± 0.028
HN-7	1.509 ± 0.020
HN-8	1.434 ± 0.047

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Cytotoxicity of Aβ species upon incubation with Ir(III) complexes

The neurotoxicity of the Ir(III) complexes and their ability to alleviate the Aβ-induced neurotoxicity was investigated using mouse neuroblastoma N2a cells (American Type Culture Collection CCL-131™). First, we examined the toxicity of all Ir(III) complexes at various concentrations ranging from 2 to 20 μM (Fig. 6a). Except for HN-1, all other iridium(III) complexes show very low cytotoxicity up to 20 μM. Then, the N2a cells were treated with Aβ₄₂ in the absence or presence of the Ir(III) complexes to investigate whether the inhibition of Aβ₄₂ aggregation can result in the rescue of Aβ₄₂ neurotoxicity (Fig. 6b). Co-treatment with any of the eight Ir(III) complexes showed a significant rescue of Aβ-induced neurotoxicity. For comparison, the N2a cells were also treated with Aβ₄₂ in the presence of curcumin, showing that curcumin dramatically increased the Aβ-induced cytotoxicity, instead of rescuing the cells (Fig. S11†).⁵⁰ Since curcumins are quite unstable in PBS and cell media,⁵⁷ the metabolites may promote Aβ-mediated cytotoxicity. However, in case of the curcumin–Ir(III) complex HN-8, the chelation between the metal center and the ligand could stabilize the curcumin structure or influence the mechanism of degradation, thus resulting in improved cell viability. Overall, the cell toxicity data reveals that Ir(III) complexes developed herein can efficiently decrease the Aβ-induced cytotoxicity in N2a cells, and thus can be employed in in vivo studies.

Fig. 6 (a) Cytotoxicity studies of Ir(III) complexes. (b) Cytotoxicity studies of Ir(III) complexes with Aβ₄₂.

In vivo blood–brain barrier permeability

The BBB permeability of the metal complexes has significant implications for in vivo applications in AD research. Until recently, few metal complexes have shown the ability to cross the BBB without assistance.^{22,27,37,58,59} To evaluate the BBB permeability of the Ir(III) complexes, we have administered daily the Ir(III) complexes with the highest log D values, HN-2, HN-3, and HN-8, as well as the complex with the highest aqueous solubility, HN-1, to 11-month old 5xFAD mice at a dose of 1 mg kg⁻¹ of body weight via intraperitoneal injection for 10 days. Excitingly, upon brain extraction and analysis by fluorescence imaging of the resulting brain sections, all three complexes show appreciably accumulation in the 5xFAD mouse brains and fluorescence staining signals in both the DAPI and GFP channels for complexes HN-2, HN-3, and HN-8 while HN-1 exhibited signal only in the DAPI channel (Fig. S12†).⁵⁰ These fluorescence imaging studies demonstrate that the developed Ir(III) complexes can successfully cross the BBB, and the in vivo accumulated Ir(III) complexes have significant colocalization with the amyloid species that were subsequently immunostained with the HJ3.4 antibody (Fig. 7), confirming the specific binding of these Ir(III) complexes to the amyloid aggregates in vivo. Considering that the lower log D, more hydrophilic complex HN-1, as well as the higher log D, more lipophilic complexes HN-2, HN-3, and HN-8 exhibit BBB penetrability, these results lend promise to the development of optimal complexes that balance these two properties for improved BBB permeability.

Fig. 7 Representative fluorescence microscopy images of brain sections from 11-month old 5xFAD mice administrated with Ir(III) complexes for 10 days. The brain sections were subsequently immunostained with the HJ3.4 antibody ([HJ3.4] = 1 μg ml⁻¹, scale bar = 125 μm) with AF-594 fluorescent tag.

Conclusions

Herein, a series of inert Ir(III) complexes HN-1 to HN-8 were designed and synthesized, and they exhibit appreciable Aβ inhibition ability in vitro and in living cells. A charged Ir(III) complex can induce a significant fluorescence increase upon binding to Aβ fibrils and Aβ oligomers, while neutral Ir(III) complexes induced a much smaller fluorescence increase despite similar affinity to fibrils. The measured log D values of the neutral complexes suggest these compounds have enhanced blood–brain barrier (BBB) permeability. Moreover, in vivo studies show that complexes HN-1, HN-2, HN-3, and HN-8 successfully penetrate the BBB and stain the amyloid plaques in AD mouse brains after a 10-day treatment, suggesting that these Ir(III) complexes could act as lead compounds for AD therapeutic and diagnostic agent development. Further ligand design to optimize the aqueous solubility while maintaining the BBB permeability of such Ir(III) complexes may produce promising lead compounds with optimal drug-like characteristics.

Data availability

The data supporting this article have been included as part of the ESI.†

Crystallographic data for HN-1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 2070088.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

L. M. M. acknowledges research funding from the NIH (R01GM114588 and RF1AG083937).

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Footnotes

† Electronic supplementary information (ESI) available: Experimental section and Fig. S1–S30. CCDC 2070088. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4dt01691b

‡ These authors contributed equally to this work.

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