Synthesis and biological evaluation of selective histone deacetylase 6 inhibitors as multifunctional agents against Alzheimer's disease

https://doi.org/10.1016/j.ejmech.2021.113821Get rights and content

Highlights

  • A high potent and isoform-selective HDAC6 inhibitor W5 was identified.

  • W5 targets multi-facets of Alzheimer's disease.

  • W5 inhibits tau hyperphosphorylation.

  • W5 inhibits Cu-Aβ aggregation and promotes Cu-Aβ aggregate disassociation.

  • W5 has good neuroprotective ability and promotes neuronal neurite outgrowth.

Abstract

Histone deacetylase 6 (HDAC6) is a potential target for Alzheimer's disease (AD). In this study, a series of novel phenothiazine-, memantine-, and 1,2,3,4-tetrahydro-γ-carboline-based HDAC6 inhibitors with a variety of linker moieties were designed and synthesized. As a hydrochloride salt, the phenothiazine-based hydroxamic acid W5 with a pyridyl-containing linker motif was identified as a high potent and selective HDAC6 inhibitor. It inhibited HDAC6 with an IC50 of 2.54 nM and was more than 290- to 3300-fold selective over other HDAC isoforms. In SH-SY5Y cells, W5 dose-dependently increased the acetylated α-tubulin levels and reduced the hyperphosphorylated tau proteins at Ser396. As an effective metal chelator, W5 inhibited Cu2+-induced Aβ1–42 aggregation and disaggregated Cu2+-Aβ1−42 oligomers, and showed protective effects on the SH-SY5Y cells against Aβ1–42- as well as Cu2+-Aβ1–42 induced cell damages, serving as a potential ligand to target AD metal dyshomeostasis. Moreover, W5 promoted the differentiated neuronal neurite outgrowth, increased the mRNA expression of the recognized neurogenesis markers, GAP43, N-myc, and MAP-2. Therefore, W5 might be a good lead for the development of novel HDAC6 inhibitors targeting multi-facets of AD.

Introduction

Alzheimer's disease (AD) is an irreversible progressive neurodegenerative disorder and the most common dementia featured with memory loss, cognitive impairments and behavioral disturbance [1]. Though the exact etiology remains unclear, AD is characterized by the accumulation of β-amyloid peptides (Aβ) and protein tau [[2], [3]]. Histone deacetylase 6 (HDAC6), a class IIB HDAC isoenzyme, is unique in its structural and physiological functions, because it signals cells through both epigenetic and non-epigenetic mechanisms and is involved in multiple cellular pathways associated with cancers, neurodegenerative diseases, rare diseases, and immunological disorders [[4], [5], [6], [7], [8]]. Increased levels and activity of HDAC6 were observed in the brains of AD patients [[9], [10]]. Due to its cytoplasmic localization, HDAC6 specifically deacetylates a variety of non-histone substrates and proteins, including α-tubulin, heat shock protein 90 (HSP90), peroxiredoxin, cortactin, survivin, β-catenin, protein tau, ubiquitin, among many others [[8], [11], [12], [13], [14], [15], [16], [17], [18]]. Higher level of acetylated α-tubulin (Ac-α-tubulin) enhances the binding of the motor protein kinesin 1 to tubulin that favors the transport of cargo proteins along the microtubule and facilitates the cleavage of the damaged organelles or misfolded proteins in the synaptic regions [[19], [20]]. The HDAC6 knockdown or inhibition increases the acetylated HSP90 that reduces the HSP90-HDAC6 binding, prompting the expression of cellular chaperone and triggering the tau degradation [[21], [22]]. HDAC6 interacts with tau and regulates tau phosphorylation as well as accumulation, and therefore, plays important roles in mediating the endogenous neuritic tau pathology [[16], [23], [24]].The inhibition of HDAC6 increases the acetylated Prx1 and Prx2 and modulates the intracellular redox status that reduces the reactive oxygen species (ROS) production and rescues the mitochondrial axonal transport impaired by Aβ [[13], [25], [26]]. Growing evidence indicates that inhibition of HDAC6 could effectively restore the impaired α-tubulin acetylation, rescue the mitochondrial transport deficits, degrade the protein aggregates, and prevent neuronal oxidative stress (Fig. 1), suggesting HDAC6 as a potential target for the treatment of neurodegenerative diseases [[8], [27], [28], [29], [30], [31], [32], [33], [34]].

In recent years, a number of selective HDAC6 inhibitors (Fig. 2) have been reported that displayed neuro-protective activity and cognitive performance improving ability in AD animal models [[35], [36], [37], [38], [39], [40], [41], [42]].

Like other zinc-dependent HDACs, the active site of HDAC6 has three main ligand binding domains: a surface binding domain, a hydrophobic channel, and a catalytic zinc-binding domain. As demonstrated by the X-ray crystal structures of HDAC6−inhibitor complexes, the typical pharmacophore model of the HDAC6 inhibitor contains a surface binding group (Cap), a zinc binding group (ZBG), and a hydrocarbon linking motif (Linker) that connects the Cap and the ZBG [[33], [34]]. In comparison with other HDACs, the HDAC6 active site cleft is slightly wider and shallower that favors compounds with larger (bulky and steric), extended caps and shorter linkers [[29], [30], [31], [32], [33], [34]]. Like the 1,2,3,4-tetrahydro-γ-carboline derivative tubastatin A (1), the phenothiazine- and memantine-based hydroxamic acids with a benzylic linker were reported as selective HDAC6 inhibitors [[43], [44], [45]]. Therefore, in the design of novel HDAC6 inhibitors for the AD therapy, we first used the phenothiazine as the Cap moiety, the cinnamenyl-, phenylethyl-, pyridyl-, oxazole-, and pyrazle-containing motifs as the possible Linker, and the hydroxamic acid as the preferred ZBG due to its strong Zn2+ ion chelating ability that is not only essential for the HDAC6 inhibitory activity, but also as effective metal chelators to target AD metal dyshomeostasis [[46], [47], [48], [49], [50]]. For a comparison, the amide functional group was also investigated as the possible ZBG. With the suitable Linker motif screened, we next examined the bulky and steric memantine and 1,2,3,4-tetrahydro-γ-carboline derivatives as the Cap moiety. Memantine hydrochloride is a N-methyl-d-aspartate receptor (NMDAR) antagonist that is widely used in clinic for the treatment of AD. The memantine pharmacophore is applied in the design of multitarget agents for AD [[45], [51]]. The 1,2,3,4-tetrahydro-γ-carboline derivative, dimebon dihydrochloride, is a histaminergic receptor antagonist that improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model, but fails in clinical phase 3 trials for AD treatment [52]. Therefore, the memantine- and 1,2,3,4-tetrahydro-γ-carboline-based HDAC6 inhibitors may act as dual or multi-targeted agents against AD. Herein, we report the synthesis and evaluation of these compounds as potential HDAC6 inhibitors targeting the multi-facet of AD.

Section snippets

Chemistry

The synthesis of the phenothiazine-based hydroxamic acids or amides was shown in Scheme 1. Phenothiazine was first treated with lithium hexamethyldisilazide (LHMDS) at −20 °C in DMF and then reacted with (E)-methyl 3-(4-bromomethylphenyl)acrylate, methyl 2-chloromethyl-oxazole-4-carboxylate, methyl 6-(bromomethyl)nicotinate, and ethyl 2-(4-chloromethyl-1H-pyrazol-1-yl)acetate to give the corresponding esters 9, 1114 in 74%, 61%, 71%, 69%, and 56% yield, respectively. The catalytic

Conclusion

A series of phenothiazine-based HDAC6 inhibitors with different Linker motifs were synthesized and evaluated for their HDAC6 inhibitory activities. The pyridyl-containing moiety was identified as a suitable linker motif, and the resulting phenothiazine-based hydroxamic W5 exhibited excellent HDAC6 inhibitory activity and isoforms selectivity in vitro and significantly increased the acetylated α-tubulin level in SH-SY5Y cells. At the concentration of 20 μM, W5 effectively reduced the

Chemistry

Common reagents and solvents were purchased from commercial suppliers and used without further purification. For all the examined compounds, 1H and 13C NMR spectra were collected on a Bruker-400 NMR or a Bruker-600 NMR instrument in deuterated solvents (DMSO‑d6, CDCl3). Chemical shifts are expressed in ppm relative to DMSO‑d6 or CDCl3. Uncorrected melting points were measured using an X-6 micromelting point apparatus (Beijing Tech. Co., Ltd). MS spectra data were obtained using an API 4000

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was partially supported by the key research and development program of Shandong province (2019GSF108045).

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