Synthesis and biological evaluation of 3-arylcoumarins as potential anti-Alzheimer's disease agents

Abstract Alzheimer's disease, a neurodegenerative illness, has the extremely complex pathogenesis. Accumulating evidence indicates there is a close relationship between several enzymes and Alzheimer's disease. Various substituted 3-arylcoumarin derivatives were synthesised, and their in vitro activity, including cholinesterase inhibitory activity, monoamine oxidase inhibitory activity, and antioxidant activity were investigated. Most of the compounds exhibited high activity; therefore 3-arylcoumarin compounds have the potential as drug candidates for the treatment of Alzheimer's disease.


Introduction
Alzheimer's disease (AD) has become one of the major diseases that threatening the health of elder people in modern society. Alzheimer's disease, a chronic progressive neurodegenerative disease, usually starts slowly and worsens over time. The most common early symptom is difficulty in remembering recent events. As the disease advances, symptoms can include problems with language, disorientation, loss of motivation, not managing and so on 1 . Gradually, bodily functions are lost, ultimately leading to death. AD is a refractory disease with complicated etiopathogenesis. Several hypotheses, such as cholinergic hypothesis, were proposed to explain the cause of the disease. The most currently available drug therapies were based on the cholinergic hypothesis, which proposes that AD is caused by reduced synthesis of the neurotransmitter acetylcholine (ACh) 2 .
The potential of acetylcholinesterase (AChE) inhibitors has been well explored and developed as an anti-AD drug 3 . There are currently five drugs used to treat AD cognitive problems, four of which are AChE inhibitors (tacrine, donepezil, galantamine, rivastigmine). The AChE activity of AD patients decreased, but butyrylcholinesterase (BuChE) activity was stable or even increased; the ratio of AChE to BuChE changed, indicating that BuChE may act as a compensatory mechanism for ACh metabolism. Studies have shown that selective BuChE inhibitors are beneficial for cognitive dysfunction of AD, and dual AChE/BuChE inhibition can maximise therapeutic efficacy 4 . Monoamine oxidase (MAO) is one of several enzymes that cause oxidative stress and dementia behaviour and psychological symptoms in AD. MAO-A inhibitors can be used as antidepressants and anxiolytics, while MAO-B inhibitors can be used alone or in combination to treat AD and Parkinson's disease 5 . Accumulating evidence shows that cholinesterase (ChE) and MAO are closely related to the disease symptoms and progression of AD. Many studies have identified the role of various ChE/MAO inhibitors in AD, while some showing positive results in clinical trials 6 .
3-Arylcoumarin refers to a class of compounds having a coumarin skeleton and having an aryl structure at the 3-position, which is rich in biological activity such as antioxidant 7 , anti-inflammatory 8 , antitumour 9 , antiviral 10 , antifungal 11 , antidiabetic 12 , MAO inhibitory activity 13,14 , ChE inhibitory activity 15 and so on. The aetiology of Alzheimer's disease is complex, and for this multi-faceted disease, the efficacy of multi-targeted drugs is better than that of single-targeted drugs. Coumarin compounds, as dual inhibitors, are promising compounds that control AD [16][17][18][19][20] . Patil et al. 21 reviewed the synthesis and designed aspects of coumarin derivatives as MAO inhibitors for AD, in which most 3-arylcoumarin compounds selectively inhibit MAO-B and give some guidance in modifying the structure. Wang et al. 15 designed, synthesised, and evaluated a series of 6-substituted 3-arylcoumarin derivatives as dual AChE/MAO-B dual inhibitors for the treatment of AD, which provided meaningful information for further development of multifunctional drugs for AD treatment. Previous studies 15,[21][22][23][24] have designed coumarin derivatives as potential inhibitors of MAO and ChE, and have achieved good results. These results encourage us to further explore the potential of coumarins as candidates for the treatment of AD.

Animals
The AB wild-type zebrafish was provided by Key Laboratory for Drug Screening Technology of Shandong Academy of Sciences. The zebrafish were cultured in an environment with a cycle of 14 h light and 10 h darkness, a pH of about 7.0, and a temperature of about 28 C. The healthy zebrafish were mated in a tank one day before the experiment, with a male to female ratio of 1:1. The separator was taken the next day and the fertilised eggs were collected 0.5 h later. The fertilised eggs were washed 3 times with aquaculture water, then disinfected with methylene blue solution, and transferred to clean culture water of about 28 C for light-control culture. Wistar

In vitro cholinesterase inhibitory activity
The anticholinesterase activity of the 3-arylcoumarin compounds was determined by the method of Ellman et al. 25 with slight modifications. AChE inhibitory activities were determined by AChE from electric eel (Macklin). BuChE inhibitory activities were determined by BuChE from horse serum (Aladdin). To a 10 ml tube, 2.65 ml of phosphate buffer solution (0.1 M, pH ¼ 8.0), 100 mL of 5,5-dithiobis-2-nitrobenzoic acid (0.75 mM in 0.1 M phosphate buffer solution, pH ¼ 8.0) were added sequentially, 50 mL AChE solution (0.2 U/mL in 0.1 M phosphate buffer solution, pH ¼ 8.0), 100 mL of different concentrations of the sample solution, shaken well, and incubated at 37 C for 5 min. Then 100 mL of substrate (1.5 mM in 0.1 M phosphate buffer solution, pH ¼ 8.0) were added, shaken well, and incubated at 37 C for 20 min. After the reaction was completed, 1 ml of sodium lauryl sulphate (4%, SDS in water) was added. The absorbance at 412 nm of the samples was measured using a spectrophotometer, and the inhibition rate of cholinesterase and the IC 50 value of each sample were calculated according to the formula. Determination of the inhibitory activity of BuChE is similar. The sample blank group replaced the substrate with PBS, and the blank group replaced the sample solution with a solvent. The sample solution was set to five concentration gradients and the experiment was repeated three times. Donepezil was used as a positive control.
where A 0 is the absorbance of blank group; A 1 is the absorbance of sample group; A 2 is the absorbance of sample blank group.

In vitro monoamine oxidase inhibitory activity
The MAO inhibitory activity of the 3-arylcoumarin compounds was determined by the method of Holt et al. 26 with slight modifications. The crude enzyme was extracted from the liver of 200-250 g of Wistar rats according to literature methods [26][27][28] where A C is the absorbance of control group; A B is the absorbance of blank group; A S is the absorbance of sample group; A SB is the absorbance of sample blank group.

In vitro antioxidant activity
The total antioxidant capacity of the 3-arylcoumarin compounds was measured by the FRAP (the Ferric Reducing Ability of Plasma) assay of Benzie et al. 29 with slight modifications. This method is based on the reduction of colourless Fe(III)-TPTZ(2,4,6-Tris(2-pyridyl)-s-triazine) complex to coloured Fe(II)-TPTZ complex by the compounds. FRAP working solution (300 mmol/L acetate buffer, 10 mmol/L TPTZ, 20 mmol/LFeCl 3 ) was ready to use. A hundred microlitres of the sample solution was added to 3 ml of FRAP reagent and then incubated at 37 C for 15 min. The experiments were repeated for three times. The absorbance was measured at 593 nm to clarify the changes. The standard curve was drawn with FeSO 4 as standard material, and the regression equation was obtained. With 1.0 mmol/L FeSO 4 as standard, the antioxidant activity of the sample is expressed in millimoles of Fe 2 SO 4 required to achieve the same absorbance.

Zebrafish behavioural experiment
The experimental sample group and the blank control group were set in a 48-well plate, and 0.5 ml of aquaculture water and a juvenile fish that developed to 72 hpf (hours post fertilisation) were added to each well. Eight juvenile fish were set up for each experimental group. Compounds 2, 20, 22 were set at four different concentrations of 10 mg/ml, 50 mg/ml, 100 mg/ml, and 1000 mg/ ml. The 48-well plate was placed in the dark box of the zebrafish behavioural analysis system. The fish were adapted to the environment for 10 min before the experiment. The zeblab software (Viewpoint, Lyon, France) was used to collect the trajectories of the juveniles in each group within 30 min, recorded every 5 min, and the total parade distance and parade time were exported by software. The average distance of each group of fish parades was calculated.

Statistical analysis
Data were shown as mean ± SD Differences between individual groups were analysed by using ANOVA followed by Dunnett's test. A difference with a p value of <.05 was considered to be significant.

Chemistry
The synthetic methods and chemicals previously reported by our research group were used 30 . The synthetic route was summarised in Scheme 1. The substituted phenylacetic acid is synthesised by substituting acetophenone, then the 3-arylcoumarin structure skeleton is synthesised by Perkin reaction with substituted salicylaldehyde under the action of triethylamine and acetic anhydride, and the target compound is obtained by acid hydrolysis (Table 1). Details on the chemical and spectroscopic characterisations of compounds were described in the references.

In vitro cholinesterase inhibitory activity
AChE is associated with neurons and axons, and is primarily responsible for ACh hydrolysis and termination. Studies have shown that long-term AChE inhibitor treatment can prevent the progression of cognitive dysfunction in patients with AD [31][32][33] . In the AD brain, BuChE may play a beneficial role by restoring cholinergic activity and/or by restoring AChE/BuChE activity ratio 34 . The AChE/BuChE inhibitory activity of all compounds was determined by the method of Ellman 25 , and donepezil was used as a reference compound in this assay. All the compounds were tested AChE/BuChE inhibitory activity in vitro, as shown in Table 2, most compounds presented inhibitory activity for AChE/BuChE. Most of the compounds exhibited moderate AChE inhibitory activity.
Notably, compound 22 (IC 50 ¼3.04 ± 0.32 mM) had relatively strong activity, which displayed weaker capacity than donepezil, and compound 20 exhibited selective AChE inhibitory activity. Only compound 7 (IC 50 ¼2.76 ± 0.57 mM) had stronger BuChE activity than donepezil, and compounds 2, 5, 10 exhibited selective BuChE inhibitory activity. The results showed that 3-arylcoumarins with a R 5 , R 6 -dihydroxy group has a significantly stronger inhibitory activity than the R 6 -or R 5 -position mono-substituted 3-arylcoumarin, and 3-arylcoumarin having a R 5 , R 6 -dihydroxy group all showed high AChE inhibitory activity. However, there is no significant tendency for the inhibitory activity of BuChE.

3.2.2.
In vitro monoamine oxidase inhibitory activity MAO plays an important role in neurotransmitter inactivation, and MAO dysfunction is considered to be the cause of many mental and neurological diseases. MAO plays an important role in AD pathology, and the progress of AD has a close correlation with MAO activity 35,36 . All the synthesised compounds were evaluated for their MAO inhibitory activity in the way of Holt 26

In vitro antioxidant activity
Oxidative damage is involved in the pathogenesis of neuronal degeneration in AD, which may represent a potential therapeutic target for slowing the progression of AD or possibly preventing the onset of AD 37 . Oxidative stress is a key feature in determining AD. All the synthesised compounds were evaluated for their antioxidant activities by means of the Ferric Reducing Ability of Plasma. Vitamin C was used as a reference in this assay. The standard curve was drawn with FeSO 4 as standard material, and the antioxidant capacity of the sample is expressed as FRAP value. As shown in experiments. In the range of 10-100 mg/ml, the total distance of zebrafish movement increases with the concentration of the compound increases. However, the high doses of compounds might cut down the movement distance of zebrafish (Figures 1 and 2).

Conclusions
We continue to study the pharmacological activities of 3-arylcoumarins synthesised by our research group. All the synthesised compounds were evaluated for their antioxidant activities in the way of the Ferric Reducing Ability of Plasma. Most compounds demonstrated moderate to high activity, among which compound 22 with several hydroxyl groups showed an excellent activity in this aspect. According to in vitro ChE inhibitory and MAO inhibitory test results, we selected selective AChE inhibitory compound 20, selective BuChE inhibitory compound 2, and dual ChE and MAO inhibitory compound 22 to study their anti-AD activity in vivo. Notably, compound 22 showed a certain effect in vivo experiments. Multi-target anti-AD compounds can modulate multiple signalling pathways or targets associated with AD, potentially producing significant clinical effects.

Ethical statement
All experiments involving living animals and their care were performed in strict accordance with the National Care and Use of Laboratory Animals by the National Animal Research Authority (China) and guidelines of Animal Care and Use issued by the University of Jinan Institutional Animal Care and Use Committee. The experiments were approved by the Institutional Animal Care and Use Committee of the School of Medicine and Life Sciences, University of Jinan. All efforts were made to minimise animal's suffering and to reduce the number of animals used.

Disclosure statement
No potential conflict of interest was reported by the authors.