Novel sampangine derivatives as potent inhibitors of Cu²⁺-mediated amyloid-β protein aggregation, oxidative stress and inflammation

Highlights

• The new derivatives exhibited high chelating potency and selectivity for Cu²⁺.

• Derivative 11 mitigated Cu²⁺-Aβ induced toxicity in Aβ42 transgenic C. elegans.

• Derivative 11 inhibited the production of ROS catalyzed by Cu²⁺ in Aβ42 transgenic C. elegans.

• Derivative 11 inhibited the expressions of pro-inflammatory cytokines induced by Cu²⁺ + Aβ_1–42 in BV2 microglial cells.

Abstract

A series of 11-substituted sampangine derivatives have been designed, synthesized, and tested for their ability to inhibit cholinesterase. Their chelating ability and selectivity for Cu²⁺ over other biologically relevant metal ions were demonstrated by isothermal titration calorimetry. Their blood-brain barrier permeability was also tested by parallel artificial membrane permeation assay. Among the synthesized derivatives, compound 11 with the strong anti-acetylcholinesterase activity, high blood-brain barrier penetration ability and high binding affinity to Cu²⁺ was selected for further research. Western blotting analysis, transmission electron microscopy, DCFH-DA assay and paralysis experiment indicated that compound 11 suppressed the formation of Cu²⁺-Aβ complexes, alleviated the Cu²⁺ induced neurotoxicity and inhibited the production of ROS catalyzed by Cu²⁺ in Aβ42 transgenic C. elegans. Moreover, compound 11 also inhibited the expressions of proinflammatory cytokines, such as NO, TNF-α, IL-6 and IL-1β, induced by Cu²⁺ + Aβ_1–42 in BV2 microglial cells. In general, this work provided new insights into the design and development of potent metal-chelating agents for AD treatment.

Introduction

“Metal Hypothesis of Alzheimer's Disease” has suggested that the neuropathogenic effects of Aβ in Alzheimer's Disease (AD) are promoted by Aβ-metal interactions [[1], [2], [3]]. These metal ions include Cu²⁺, Zn²⁺ and Fe³⁺. Among them, Cu²⁺ plays a key role in the pathogenesis of AD. On the one hand, Cu²⁺ trigger and accelerate the converting of the soluble monomers of Aβ to neurotoxic Aβ oligomers [4,5]. On the other hand, the complexation of Cu²⁺ with Aβ is responsible for the reactive oxygen species (ROS) related neurotoxicity. H₂O₂ catalyzed by Cu²⁺-Aβ complex from O₂ and substrates, like ascorbic acid and cholesterol, is the key source of the oxidative stress observed in AD [6]. In addition, recent studies have also shown that Cu²⁺ exacerbated Aβ-elicited proinflammatory cytokines (such as IL-1β, TNF-α, and IL-6) release in microglia [7,8]. Elevated levels of proinflammatory cytokines were also capable of significantly reducing the expression of low-density lipoprotein-related receptor protein-1 (LRP1) in human microvascular endothelial cells in a concentration-dependent manner [7,9]. This proinflammatory cytokine-mediated downregulation of LRP1 consequently reduced LRP1 transcytosis-mediated clearance of Aβ [9,10]. Thus, the pathological impact of Cu²⁺ on inflammatory responses have served as key mechanisms to explain, in part, the Cu²⁺ exposure as an environmental risk factor for AD.

Thus, the restoration of Cu²⁺ homeostasis by using specific chelators has been considered a key pharmacological target [11]. Therefore, the application of metal ions chelators with high selectivity for Cu²⁺, high blood-brain barrier (BBB) permeation and no interference to the normal metabolism of the metal ions has been widely studied for ameliorating pathologies and the cognitive functions in AD [12,13]. These metal-protein attenuating compounds, such as clioquinol (CQ) and PBT2 (Fig. 1), are reported to have the highest interest in AD treatment. Unfortunately, CQ and PBT2 entered anti-AD clinical trials but failed due to their neurotoxicity and weak chelating efficacy and selectivity to Cu²⁺ [14].

The copyrine alkaloid sampangine (Fig. 1) belongs to the aporphine family of alkaloids, which was widely distributed in Annonaceae plants [[15], [16], [17], [18]]. Plant-derived sampangine alkaloid shows broad and potent antibacterial [[19], [20], [21], [22]], antifungal [16,[23], [24], [25], [26], [27]], antimalaria [17], anti-inflammatory [28] and antiphrastic [29]. In our previous studies, 4-aminoalkylaminosampangine derivatives had good BBB penetration ability [30]. Moreover, one of these derivatives SD-1 (Fig. 1) was able to modulate Cu²⁺-mediated multiple pathological elements in AD [31]. The reason may be that the relative position of the nitrogen atom and oxygen atom in sampangine is very similar to representative metal chelators (such as CQ and PBT2). It suggested that sampangine should have good metal chelating ability.

To add Cu²⁺ binding sites and enhance the chelating potency and selectivity of chelators, in this paper, we designed a series of 11- substituted sampangine derivatives (Fig. 1). We reported that 11- substituted sampangine derivatives were capable of regulating Cu²⁺-induced Aβ aggregation, and its associated neurotoxicity as well as oxidative stress and inflammation mediated by Cu²⁺ were also discussed. Moreover, these derivatives showed high BBB penetration ability and strong inhibitory potency toward acetylcholinesterase. Additionally, the chelating ability and the specificity of derivatives for Cu²⁺ and the other metal ions (such as K⁺, Ca²⁺, and Mg²⁺), which play an important role in maintaining the normal physiological functions of organisms, were validated by the isothermal titration calorimetry (ITC). Moreover, one of synthesized compound 11 was capable of regulating Cu²⁺-induced Aβ aggregation and its associated neurotoxicity as well as oxidative stress and inflammatory response mediated by Cu²⁺. Taken together, these results demonstrated that 11- substituted sampangine derivatives were multifunctional inhibitors of Cu²⁺-mediated Aβ aggregation, which could be essential for their noticeable modulating reactivities toward multiple pathological features found in AD.