Design, synthesis, and biological evaluation of novel 2-acetylphenol-rivastigmine hybrids as potential multifunctional agents for the treatment of Alzheimer’s disease

Herein, a series of 2-acetylphenol-rivastigmine hybrids were rationally designed as multifunctional agents for the treatment of AD through multi-target-directed ligands (MTDLs) strategy. The biological activity in vitro showed that compound 3c was a pseudo-irreversible and selective huAChE inhibitor (IC50 = 8.7 μM). Compound 3c was also a significant antioxidant with ORAC value of 1.2 eq. Moreover, compound 3c showed potent MAO-B inhibitory activity with IC50 values of 2.9 μM. In addition, compound 3c displayed selective metal chelation property. Furthermore, compound 3c showed significant neuroprotective effect on H2O2-induced PC12 cells injury. More importantly, compound 3c displayed favorable drug-like property and good blood brain barrier permeability in vitro. Thus, compound 3c was a promising multifunctional agent in vitro for the treatment of AD, deserving for further investigations. Graphical abstract Graphical abstract


Introduction
Alzheimer's disease (AD) is an age-related neurological disorder and characterized by memory loss, decline in thinking ability, language skills, severe behavioral anomalies and eventual death. This disease affects over 55 million people worldwide, which has been a serious challenge to the public health system [1,2]. None of the available drugs approved by the FDA slows, halt or reverse the progression of AD. They just temporarily present modest symptomatic effects [3]. Thus, the development of disease-modifying drugs is a great unmet medical need for the AD patients.
Due to the multifactorial nature of AD, the success of a therapeutic approach is likely to depend on the simultaneous modulation of more than one AD-relevant target, These authors contributed equally: Cong Deng, Jing Mi * Zhipei Sang sangzhipei@126.com * Hui Li lh7202@sina.com which leads to a new paradigm in drug discovery for AD, namely the multi-target-directed ligands (MTDLs). MTDLs can hit two or more AD-relevant complementary targets and produce synergistic effect on the disease network by improving clinical outcomes [4][5][6]. ChEs inhibitors have presented significant improvement of cognitive impairment, such as donepezil, tacrine, rivastigmine and galantamine. Therefore, the MTDLs involving ChEs inhibition have drawn great attention and have been developed by many groups, and some promising multifunctional candidates are in progress [7][8][9]. In addition, monoamine oxidases (MAOs), the important factors involved in oxidative stress, have attracted more and more attention. MAOs are flavin adenine dinucleotide (FAD)-containing enzymes, including MAO-A and MAO-B. They are responsible for the regulation and metabolism of major monoamine neurotransmitters and xenobiotic amine [10]. Accumulation of evidences reveal that levels of MAO-B activity could increase up to 3-fold in the temporal, parietal and frontal cortex of AD patients, the high expression levels of MAO-B in neuronal tissue can increase the level of oxidative free radicals, and further accelerate the former of Aβ plaques [11]. Therefore, selective MAO-B inhibitor would be a promising strategy to treat AD. Such as, the selective MAO-B inhibitor rasagiline, has been carried out a phase 2 trial in people with mild-to-moderate AD, and it presents trends to better performance [12].
On the other hand, the oxidative stress hypothesis states that the increase of production of reactive oxygen species (ROS) and free radicals leads to oxidative damage, which acts as central processes in AD pathogenesis [13,14]. Moreover, the high levels (Cu 2+ , Fe 2+ , and Zn 2+ ) in the AD brain cause oxidative stress and further increase the production of ROS. More importantly, Cu 2+ , Zn 2+ and other disordered metal ions also accelerates the aggregation of Aβ [15]. Thus, compound with antioxidant and biometal chelating property may be a potential approach for the treatment of AD.
Recent study showed that 2-acetylphenols are promising leads for the design of MAO-B inhibitors, particularly, DDDT-2d is a high selective and effective MAO-B inhibitor (IC 50 = 2.9 nM) [16]. In addition, many investigations suggest that compounds having an hydroxyl group and a ketonic group would undergo chelation easily with a formation of six membered ring [17][18][19]. So, 2-acetylphenol is a promising skeleton for the development of multifunctional agents. Rivastigmine is a dual AChE/BuChE inhibitor approved by FDA for the treatment of mild to moderate AD. The N-ethyl-N-methylcarbamoyl moiety is the key pharmacophore, which has been widely used and developed to obtain novel ChEs inhibitors by our group [18][19][20] and other groups [21,22]. Herein, we plan to introduce different carbamate moiety into the promising 2-acetylphenol skeleton to obtain novel 2-acetylphenol-rivastigmine hybrids by MTDLs strategy (Fig. 1). The synthesized compounds were

Molecular modeling study of AChE with 3c
The docking program was employed to explore possible binding mechanism of 3c with huAChE ((PDB code: 4ey4)) using AutoDock 4.2 package with Discovery Studio 2.5 [20]. Figure 2 shows that 3c interacted with huAChE via multi-interactions. There was one intermolecular hydrogen bonding was formed between the H atom of hydroxy group and amino acid residue Tyr341. Another intermolecular hydrogen bonding was formed between the carbonyl group of 2-acetyl fragment and residue Ser293. In addition, two intermolecular hydrogen bondings were formed between the two oxygen atom of Ocarbamate moiety and amino acid residue Phe295. Moreover, one Sigma-Pi interaction was formed between the benzene ring and residue Val294. Furthermore, some hydrophobic interactions were also observed between the ligand 3c and Trp286, Ser293, Phe295 and Tyr341. Thus, these interactions may provide reasonable explanation for its potent inhibitory potency.

The reversibility study of 3c
To determine whether compounds 3c was a reversible huAChE inhibitor, we tested the recovery of huAChE inhibitors inhibition after dilution [26]. In Fig. 3A, when donepezil and rivastigmine diluted to 0.1 × IC 50 , respectively, the huAChE activity increased to 9.3% and −3.2%, respectively, compared with IC 50 donepezil and IC 50 rivastigmine, suggesting that donepezil was a reversible huAChE inhibitor and rivastigmine might be a irreversible huAChE inhibitor. Under the same condition, the 0.1 × IC 50 tested compound 3c increased huAChE activity to 0.6%. To further investigate the reversibility of inhibition, the recovery of huAChE inhibitors inhibition after dilution was carried out with time monitoring. As shown in Fig. 3B, the 0.1 × IC 50 donepezil restored the huAChE activity to 101.6% at 60 min, and the 0.1 × IC 50 rivastigmine gradually restored the huAChE activity to 80.1% at 120 min, revealing that donepezil was a reversible huAChE inhibitor and rivastigmine was a pseudo-irreversible huAChE inhibitor. The data also showed that the 0.1 × IC 50 compound 3c restored the huAChE activity to 85.7%, which was

Antioxidant activity assay
The antioxidant potency of the target derivatives were also evaluated by the ORAC-FL method (Oxygen Radicals Absorbance Capacity by Fluorescence) and vitamin E analog Trolox was used as a standard [18,26]. The results in Table 1 showed that compounds 3a-3n with monosubstituted carbamate fragment showed better antioxidant potency (ORAC values ranging from 1.0 eq to 1.2 eq) that compounds 4a-4k with bis-substituted carbamate fragment (ORAC values ranging from 0.1 eq to 0.3 eq), suggesting that the number of hydroxy group played key role on the antioxidant activity. In addition, the carbamate fragment and substituted 2-acetyl did not produce obvious effect on antioxidant potency. The representative compounds 3c and 3j showed good antioxidant activity with ORAC values of 1.2 eq and 1.0 eq, respectively.

The inhibitory potency against MAO-A and MAO-B
To further study the multi-functional biological profiles of the synthesized derivatives, the inhibitory potency against MAO-A and MAO-B (recombinant human enzyme) was performed and using Iproniazid as positive drug [27]. As listed in Table 1

Metal chelation properties
In order to test the metal chelating property of the target compounds, representative compounds 3c and 3j were selected to test the chelating property by UV-visual spectrometry using Cu 2+ , Zn 2+ , Al 3+ and Fe [18,26]. Compound 4c was also selected to tested chelation property compared with compound 3c. Figure 4A showed that the maximum absorption wavelength shifted from 317 nm to 386 nm and 374 nm, respectively after adding CuCl 2 and AlCl 3 to the solution of 3c, respectively. While, adding FeSO 4 and ZnCl 2 to the solution of 3c, respectively, the maximum absorption at 317 nm did not produce obvious shift. The results suggested that 3c selectively chelated Cu 2+ and Al 3+ , forming the 3c-Cu 2+ and 3c-Al 3+ complex, respectively. In order to confirm the hydroxy group and carbonyl group played key role in chelaing property, compound 4c was tested under the same condition, as presented in Fig. 4B, the maximum absorption at 336 nm did not produce obvious shift after adding CuCl 2 , AlCl 3 , FeSO 4 and ZnCl 2 to the solution of 4c, respectively. The results from Fig. 4A, B showed that the hydroxy group and carbonyl group could chelate Cu 2+ and Al 3+ . The similar phenomenon was also observed in Fig. 4C, the maximum absorption wavelength shifted from 333 nm to 389 nm and 390 nm, respectively after adding CuCl 2 and AlCl 3 to the solution of 3j, respectively, while, adding FeSO 4 and ZnCl 2 to the sluction of 3j, respectively, the maximum absorption at 333 nm did not produce obvious shift, suggesting that compound 3j was also a selective metal chealtor and formed the 3c-Cu 2+ and 3c-Al 3+ complex, respectively. The stoichiometry of the 3c-Cu 2+ complex or 3j-Cu 2+ complex was determined applying molar ratio method at 392 nm by increasing amounts of CuCl 2 . Figure 5A showed that the two straight lines intersected at a mole fraction of 0.97, suggesting a 1:1 stoichiometry for complex 3c-Cu 2+ . Similarly, Fig. 5B displayed a mole fraction of 0.95, suggesting a 1:1 stoichiometry for complex 3j-Cu 2+ .

Neuroprotective effects on H 2 O 2 -induced PC12 cell injury
Based on the above, compounds 3c and 4c were selected to evaluate neuroprotective effect on H 2 O 2 -induced PC12 cell injury. Firstly, the cytotoxicity of compounds 3c and 4c were tested using MTT assay, as displayed in Fig. 6, both compounds 3c and 4c did not produce obvious cytotoxicity until the concentration increased to 40 μM. Thus, both 3c and 4c showed a good safety range at concentrations of 20 μM and below. Further, compounds 3c and 4c (5.0, 10.0, and 20.0 μM) were selected to determine the neuroprotective effect on H 2 O 2 -induced PC12 cell injury using CCK-8 assay [28]. As displayed in Fig. 7, the cell viability sharply decreased to 37.0% (p < 0.001 vs control) after treating with

Theoretical evaluation of ADME properties
Compounds 3c and 4c were selected to predict the druglike properties using the Molinspiration property program [29]. The data was shown in Table 2, compound 3c and 4c do not break any point of the Lipinski's rule of five, deserving further investigations.
In vitro blood-brain barrier permeation assay The blood-brain barrier permeability is key for the development of CNS drugs, such as anti-AD drugs. So, Fig. 6 The cytotoxicity of compound 3c and 4c on PC12 cells were tested using MTT assay Fig. 7  the representative compounds 3c and 4c were selected to investigate the BBB permeability through the parallel artificial membrane permeation assay of the blood-brain barrier (PAMPA-BBB) [20,30]. First of all, 11 reported drugs were obtained to verify this method as our previous reported, and then the ranges of permeability P e (×10 −6 cm/s) was established: when P e < 1.61, the compounds showed weak BBB permeation; when 1.61 < P e < 3.44, the compound displayed uncertain BBB permeation; when P e > 3.44, the compounds suggested high BBB permeation. The tested results were listed in Table 3, the drug testosterone with 20.7 × 10 −6 cm/s permeability could cross BBB in vitro, while norfloxacin with 0.18 × 10 −6 cm/s permeability did not cross BBB in vitro. Correspondingly, compounds 3c and 4c presented good BBB permeation with P e values of 13.1 × 10 −6 cm/s and 18.3 × 10 −6 cm/s, respectively, which was similar with testosterone, suggesting that compounds 3c and 4c could cross BBB through passive diffusion and deserving further investigation.

Conclusion
In summary, 25 novel 2-acetylphenol-rivastigmine hybrids were rationally designed as multifunctional agents for the treatment of AD through MTDLs strategy. The target compounds were synthesized and evaluated by AChE/BuChE inhibition, antioxidant activity, MAO-A/ MAO-B inhibition, metal chelation, neuroprotective effect, and BBB permeability in vitro. The results in vitro displayed that compounds 3c and 3j were significant AChE inhibitors with IC 50 values of 8.7 μM and 11.4 μM, respectively. Compound 3c and 3j also showed good antioxidant activity with ORAC value of 1.2 eq and 1.0 eq, respectively. Moreover, compounds 3c and 3j showed potent MAO-B inhibitory activity with IC 50 values of 2.9 μM and 10.5 μM, respectively. In addition, compounds 3c and 3j were selective metal chelators. Furthermore, compound 3c demonstrated significant neuroprotective effect on H 2 O 2 -induced PC12 cells injury. More importantly, compound 3c demonstrated favorable drug-like property and good blood-brain barrier permeability in vitro. Therefore, compound 3c was a promising multifunctional agent for treating AD, deserving further investigation.

Experimental
Chemistry Unless otherwise noted, all the chemicals and solvents were bought from Shanghai Titan Scientific Co., Ltd. and Sigma-Aldrich. All the reactions were monitored by thin-layer chromatography (TLC), crude products were purified by column chromatography using silica gel (230-400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. (China). The 1 H NMR and 13 C NMR spectra of target compounds were recorded using a Varian INOVA at 400 and 100 MHz, respectively. The high-resolution mass spectra were obtained using a Waters Xevo G2-XS-Qtof mass spectrometer. The purity of all final compounds was determined by high-performance liquid chromatography (HPLC) analysis to be over 97%. HPLC analysis was carried out on a Waters X-Bridge C18 column (4.6 mm × 150 mm, 5 μm) at a flow ratio of 0.8 mL/min.

Recombinant human MAO-A and MAO-B inhibition studies
Recombinant human MAO-A and MAO-B were purchased from Sigma-Aldrich and stored at −80°C. solutions of test compounds were prepared in DMSO (2.5 mM) and diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM) to a final volume of 500 μL containing various concentrations of test compounds (0-100 μM) and kynuramine (45 μM for MAO-A and 30 μM for MAO-B). The detailed procedure referenced our previous work [27].

Antioxidant activity
The oxygen radical absorbance capacity fluorescein (ORAC-FL) assay was applied to assess the antioxidant activity. The detailed procedure was reported in our previous work [18,26].

Metal chelation property
The metal chelation property was investigated by UV absorption in a Shimadzu UV-2450 spectrophotometer [18]. Briefly, the UV absorption of compound 3c, 3j, or 4c, in the absence or presence of CuCl 2 , FeSO 4 , ZnCl 2 , and AlCl 3 , was recorded with wavelength ranging from 200 to 600 nm after incubating for 30 min at room temperature. The final volume of reaction mixture was 1 mL, and the final concentrations of tested compound and metals were 37.5 μM. Numerical subtraction of the spectra of the metal alone and the compound alone from the spectra of the mixture gave the difference UV-vis spectra due to complex formation. Then, the molar ration was determined using titrating the methanol solution of tested compound with ascending of CuCl 2 . Briefly, the final concentration of tested compound was 37.5 μM, and the final concentration of Cu 2+ ranged from 7.5 to 93.75 μM. The UV spectra was recorded and treated by numerical subtraction of CuCl 2 and compound 3c and 3j at corresponding concentrations, plotted versus the mole fraction of compound 3c and 3j [17,23].
Neuroprotective effects on H 2 O 2 -induced PC12 cell injury PC12 cells were seeded into 96-well plates at a density of 5 × 10 4 cells/mL in DMEM medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO 2 at 37°C. After 24 h incubation, compounds 3c and 4c (5.0 μM, 10 μM, and 20 μM) was added and incubated with the cells for 2 h. Then, H 2 O 2 (final concentration was 500 μM) was added to the PC12 cell, after 24 h, the cell viability was measured with CCK-8 assay. The absorbance was determined at 450 nm using a microplate reader. Duncan's test method was used for statistics. Data are expressed as mean ± SD.