Design, synthesis and evaluation of resveratrol-indazole hybrids as novel monoamine oxidases inhibitors with amyloid-β aggregation inhibition

Bioorganic Chemistry 76 (2018) 130–139

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Design, synthesis and evaluation of resveratrol-indazole hybrids as novel monoamine oxidases inhibitors with amyloid-b aggregation inhibition Jin-Shuai Lan a,1, Yun Liu a,1, Jian-wei Hou a, Jing Yang a, Xin-Yu Zhang a, Yuan Zhao b, Sai-Sai Xie c,⇑, Yue Ding a,⇑, Tong Zhang b a b c

Experiment Center of Teaching & Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China

a r t i c l e

i n f o

Article history: Received 29 August 2017 Revised 12 November 2017 Accepted 15 November 2017 Available online 15 November 2017 Keywords: Monoamine oxidase Ab (1–42) aggregation Alzheimer’s disease Molecular docking

a b s t r a c t Novel hybrids with MAO and Ab (1–42) self-aggregation inhibitory activities were designed and synthesized with the employment of indazole moiety and resveratrol. The biological screening results indicated that most compounds displayed potent inhibitory activity for Ab (1–42) self-aggregation, and obvious selective inhibition to MAO-B. Among these compounds, compound 6e was the most potent inhibitor not only for hMAO-B (IC50 = 1.14 lM) but also for Ab (1–42) self-aggregation (58.9% at 20 lM). Molecular modeling and kinetic studies revealed that compound 6e was a competitive MAO-B inhibitor, which can occupy the active site of MAO-B, and interact with Ab (1–42) via p-p and cation–p stacking interactions. In addition, compound 6e had no toxicity on PC12 cells and could cross the BBB. Collectively, all these results suggested that compound 6e might be a promising multi-target lead compound worthy of further investigation. Ó 2017 Published by Elsevier Inc.

1. Introduction Alzheimer’s disease (AD), a chronic and slowly progressive disorder, is characterized by the loss of memory, learning and other cognitive functions [1,2]. At present, there are several factors including b-amyloid (Ab) deposits, low levels of acetylcholine, s-protein aggregation, inflammation, oxidative stress and dyshomeostasis of biometals have been found to play vital roles in the pathogenesis of AD [3–7]. Among these factors, the ‘‘amyloid hypothesis” states that Ab was thought to be a potential target for AD therapy, in which the aggregation of Ab, especially for the isoforms of Ab (1–40) and Ab (1–42), leads to the formation of senile plaques, resulting in neuronal dysfunction in AD patients [8,9]. Moreover, the progres-

Abbreviations: AD, Alzheimer’s disease; MAO, monoamine oxidase; CNS, central nervous system; 5-HT, 5-hydroxy-tryptamine; NE, norepinephrine; DA, dopamine; FAD, flavin adenine dinucleotide; SI, selectivity index; BBB, bloodbrain barrier; MTDL, multi-target-directed ligand; Th-T, thioflavin-T; PAMPA-BBB, parallel artificial membrane permeation assay for BBB; MTT, methyl thiazolyl tetrazolium. ⇑ Corresponding authors. E-mail addresses: [email protected] (S.-S. Xie), dingyue-2001@ hotmail.com (Y. Ding). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bioorg.2017.11.009 0045-2068/Ó 2017 Published by Elsevier Inc.

sive accumulation of Ab is accompanied by inflammation and oxidative stress, which leads to neurodegeneration further [10,11]. Besides, oxidative stress appears to be a major determinant in AD pathogenesis and progression, in which monoamine oxidases (MAOs) were regarded as the most important factors. And it is reported that MAOs can not only bind tightly to the outer mitochondrial membrane of glial, neuronal and other cells, but also can regulate and metabolize biogenic amines by oxidative deamination, such as serotonin, dopamine (DA) and epinephrine [12]. Ordinarily, there are two distinctive MAO enzymes contained in mammals, namely MAO-A and MAO-B, which were distinguished by their substrate and inhibitor selectivity [13–17]. Compared to MAO-A that preferentially metabolizes epinephrine, norepinephrine, and serotonin, and is selectively inhibited by clorgyline, MAO-B is selectively inhibited by selegiline or rasagiline, and specifically deaminates b-phenethylamine [18]. Because of the reason that MAOs could terminate the actions of neurotransmitter amines in the central nervous system (CNS), they are regarded as attractive targets in the therapy of psychiatric and neurological disorders. Besides, MAO-B increases with age and its activity is found elevated in AD patients, leading to an enhanced metabolism of dopamine and to the production of large amounts of hydrogen peroxide, which ultimately give rise to neuronal damage [19].

J.-S. Lan et al. / Bioorganic Chemistry 76 (2018) 130–139

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Fig. 1. Drug design strategy for bifunctional ligands.

Scheme 1. Syntheses of target compounds 6a–6f, 7, 8 and 9. Reagents and conditions: (a) C2H5OH, reflux, 4–12 h.

Inhibition of MAO-B can improve the symptoms of AD, and several selective MAO-B inhibitors like selegiline and rasagiline have been demonstrated to retard the further neurodegeneration in AD [20]. Currently, selective MAO-A inhibitors are widely applied in the therapy of depression [21], while selective MAO-B inhibitors have been employed alone or in combination to treat Alzheimer’s and Parkinson’s diseases [22]. Considering the complexity of AD, drugs with a single target might be insufficient in the treatment of such disease. Thus, a more appropriate therapeutic strategy based on the multi-targetdirected ligands (MTDLs) paradigm has been proposed and applied in the design of multi-target compounds against AD [23–25]. Resveratrol (trans-3, 40, 5-trihydroxystilbene), a natural phenolic compound, has been widely investigated as anti-aging, antiinflammatory, cardioprotective and anticancer agents [26–29]. Researches have been focused on its effects against AD and it has

been reported that resveratrol can inhibit Ab self-aggregation and counteract Ab toxicity by its antioxidant properties in cellular models [30]. And recently, multi-functional compounds based on resveratrol targeting Ab aggregation have been extensively studied for AD treatments [31]. Besides, indazoles and their optimized chemical structures were selected to develop new MAO inhibitors [32,33], for example, indazole-carboxamides were discovered as highly potent, selective, competitive, and reversible inhibitors of MAO-B: the most potent derivatives, N-(3,4-dichlorophenyl)-1-m ethyl-1H-indazole-5-carboxamide, were subnanomolar potency. As mentioned above, these findings suggested that both resveratrol and indazole hold a promising in the therapy of AD by targeting different key pathways of the disease pathogenesis. To continue our investigations on the potential application of natural products in the AD field [34,35], synthesis of small molecules by direct introduction of the pharmacophore moiety of MAO-B

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Table 1 hMAO inhibitory activities of the synthesized compounds.

a b c d

Compounds

R

MAO-A IC50 (lM)a

MAO-B IC50 (lM)a

Selectivity indexc

6a 6b 6c 6d 6e 6f 7 8 9 Resveratrol Iproniazid Rasagiline

H 2-OH 3-OH 3,4-di-OCH3 3,4-di-Cl 3-OCH3, 4-OH – – –

9.5 ± 0.3%b 16.4 ± 0.1%b 66.39 ± 1.08 27.1 ± 2.1%b 30.4 ± 1.2%b 9.52 ± 0.38 10.4 ± 0.5%b 18.19 ± 0.73 18.58 ± 0.59 18.63 ± 1.03 6.78 ± 0.35 0.80 ± 0.02

11.28 ± 0.53 47.9 ± 1.9%b 85.43 ± 1.97 12.61 ± 0.98 1.14 ± 0.09 25.8 ± 1.4%b 37.0 ± 0.8%b 15.36 ± 0.91 1.94 ± 0.08 31.28 ± 0.86 8.24 ± 0.18 0.041 ± 0.009

N.D.d N.D. 0.78 N.D. N.D. N.D. N.D. 1.18 9.58 0.60 0.82 19.51

– –

IC50: 50% inhibitory concentration (means ± SEM of three experiments). Test concentration is 100 lM. Selectivity index = IC50 (MAO-A)/IC50 (MAO-B). N.D. – Not detected at the concentrations tested.

inhibitors into the structure of Ab interaction was proposed in this paper (Fig. 1), which was thought can afford bifunctional compounds with MAO inhibition and Ab interactive activities. The stilbene structure was chosen as the basic structure of bifunctional small molecules considering the properties of strong binding affinity to Ab species, blood-brain barrier (BBB) penetration, and easy removal from normal brain tissue [36]. For MAO inhibition, we introduced the indazole structure to the target compounds. Based on the design introduced above, the novel resveratrolindazole hybrids were synthesized as multipotent AD modifying agents, and all products were evaluated for their biological activity including the abilities in MAO inhibition, Ab anti-aggregation. Moreover, kinetic and molecular modeling studies were performed to further explore their mechanism of interaction with MAO-B and Ab (1–42). 2. Results and discussion 2.1. Chemistry The target compounds 6a–6e, 7, 8 and 9 were efficiently synthesized as shown in Scheme 1 in good yields (80–90%) [37]. The commercially available 1H-indazol-5-amine derivatives were reacted in ethanol with one equivalent of the respective aromatic/ heteroaromatic aldehyde derivatives under reflux condition (Scheme 1). The purity of all compounds was confirmed to be higher than 95% through analytical HPLC performed with Agilent 1200 HPLC System. All synthesized compounds were elucidated by spectroscopic measurements (IR, Mass, 1H NMR and 13C NMR). In the IR spectra, skeleton vibrations for benzene rings appeared in between 1418 and 1584 cm 1. The characteristic strong bands appeared for CH = N stretching at 1600–1647 cm 1. In the 1H NMR spectra, aromatic protons appeared as set of multiplet in the region d 5.97– 8.76 ppm and CH = N protons resonated as a singlet between d 8.16 and 9.71 ppm. Moreover, in the 13C NMR spectra, the carbon resonance frequencies of the CH = N was at d 152.6–162.4 ppm.

The aromatic carbons appeared at d 111.1–160.7 ppm. Finally, the AOCH3 groups appeared at d 55.9–56.1 ppm. 2.2. Inhibition of hMAO activity For compounds 6a–6e, 7, 8, 9 and resveratrol, the MAO inhibitory activities were tested with iproniazid and rasagiline as Ref. [38]. The corresponding IC50 values with MAO and the selectivity ratios were shown in Table 1. It could be concluded that most resveratrol-indazole hybrids were moderate to good MAO inhibitors with IC50 values in the micromole range. Among the synthesized compounds, compound 6e (IC50 = 1.14 lM) was the most potent and selective inhibitor against MAO-B, as a 7.2-fold higher active was obtained when compared to iproniazid. By contrast, compound 6f exhibited the highest inhibitory activity against MAO-A (IC50 = 9.52 µM), which was similar with that of iproniazid. Initially, compound 6a was produced by introducing the indazole structure to resveratrol. The results showed that compared with resveratrol, compound 6a displayed a larger decrease in MAO-A activity and an increase in MAO-B activity, which showed that compound 6a was a selective MAO-B inhibitor. This indicated that the introduction of indazole structure might be good for MAO-B inhibition. Then, substituents with varying positions and electronic properties were introduced to phenyl ring to study the possible effects on MAO inhibition [33]. For MAO-A, compounds 6a-6e showed weak or no inhibitory activity, while compounds 6f possessing 4-OH on phenyl ring exhibited large enhancement in MAO-A inhibition (IC50 = 9.52 lM), which might suggest the 4-OH on phenyl ring was good for MAO-A inhibition. For MAO-B, among compounds 6a–6f, it was noteworthy that the compounds substituted with electron-donating groups exhibited large decrease in MAO-B inhibition. For example, compound 6c (IC50 = 85.43 lM for MAO-B) possessing an OH was about 8-fold less active than that of compound 6a. However, compounds bearing electron-withdrawing groups exhibited large enhancement in MAO-B inhibition. For example, compound 6e (IC50 = 1.14 lM for MAO-B) substituted with Cl, increased the MAO-B inhibition

J.-S. Lan et al. / Bioorganic Chemistry 76 (2018) 130–139

Fig. 2. Recovery of enzyme activity after dilution. Human MAO-B were preincubated with compound 6e at concentrations equal to 10  IC50 and 100  IC50 for 30 min and then diluted to 0.1  IC50 and 1  IC50, respectively. The residual enzyme activities were subsequently measured.

potency of compound 6a by 9.9-fold. Moreover, in order to study the possible effects on MAO inhibition, benzene ring was replaced by the different heterocycles (pyridine, furan and chromone) to study the possible effects on MAO inhibition. Compound 7 containing a six-membered heterocycle, pyridine, showed no inhibition for both MAO-A and MAO-B, whereas the furan-2-yl derivative 8 (IC50 = 18.19 lM for MAO-A, IC50 = 15.36 lM for MAO-B) not only kept MAO-B inhibitory activity but also obtained more than 5.5fold potent for MAO-A when compared to compound 6a. Surprisingly, a further augment in MAO potency and a significant selectivity for MAO-B were observed for compound 9 (IC50 = 1.94 lM for MAO-B, SI = 9.58) containing a chromone.

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Fig. 3. Kinetic study on the mechanism of human MAO-B inhibition by compound 6e. Overlaid Lineweaver-Burk reciprocal plots of the inhibition of hMAO-B in the presence of different concentrations of compound 6e with p-tyramine (0.05–1.5 mM) as substrate are shown.

2.3. Reversibility of hMAO-B inhibition As we know, there are two kinds of MAO-B inhibitors, namely, irreversible and reversible. To examine which kind the resveratrol-indazole hybrids was, the dilution assay was evaluated [39]. Compound 6e was selected as a representative inhibitor since it displayed the most potent MAO-B inhibitory activity. To investigate whether compound 6e was a reversible inhibitor of MAO-B or not, the recovery of enzymatic activity after dilution of the enzyme-inhibitor complexes was evaluated, and an irreversible

Fig. 4. (A) 3D docking model of compound 6e with hMAO-A. Atom colors: yellow-carbon atoms of compound 6e, gray-carbon atoms of residues of hMAO-A, green- FAD of hMAO-A, dark blue-nitrogen atoms, red-oxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (B) 3D schematic diagram of docking model of compound 6e with hMAO-B. (C) 2D docking model of compound 6e with hMAO-A. (D) 2D schematic diagram of docking model of compound 6e with hMAO-B.

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Table 2 Inhibition of Ab (1–42) self-induced aggregation by target compounds. Compound

R

Ab(1–42) aggregation inhibition (%)a

IC50 (lM)b

6a 6b 6c 6d 6e 6f 7 8 9 Curcumin Resveratrol

H 2-OH 3-OH 3,4-di-OCH3 3,4-di-Cl 3-OCH3, 4-OH – – – – –

56.7 ± 0.9 42.9 ± 0.7 59.8 ± 2.6 57.4 ± 3.1 58.9 ± 1.6 55.9 ± 1.1 51.2 ± 1.6 50.3 ± 0.9 61.5 ± 1.5 54.6 ± 2.6 64.2 ± 1.6

19.6 ± 0.2 23.6 ± 0.4 19.0 ± 0.7 19.3 ± 0.5 19.5 ± 0.6 19.6 ± 0.9 19.8 ± 0.5 20.1 ± 0.9 18.8 ± 1.1 19.8 ± 0.5 17.9 ± 0.2

a

Inhibition of Ab (1–42) self-induced aggregation, the thioflavin-T fluorescence method was used, the mean ± SD of at least three independent experiments and the measurements were carried out in the presence of 20 lM compounds. b IC50: 50% inhibitory concentration (means ± SEM of three experiments).

inhibitor, pargyline, was used as a reference compound. MAO-B was preincubated with compound 6e at concentrations of 0, 10 and 100⁄IC50 for 30 min and then diluted 100-fold to yield concentrations of 0, 0.1 and 1⁄IC50. For reversible inhibition, enzymatic activity is expected to be approximately 90% after dilution to 0.1⁄IC50, and 50% after dilution to 1⁄IC50. For an irreversible inhibitor, enzyme activity is expected not to recover after diluting the enzyme–inhibitor complex. From the Fig. 2, it can be seen that, after the dilution of 6e to 0.1⁄IC50, the MAO-B catalytic activities are recovered to levels of 83% of the control value (recorded in absence of inhibitor). After dilution to 1⁄IC50, the MAO-B catalytic activities are recovered to levels of 45%. This behavior is consistent with a reversible interaction of the compound with MAO-B. After similar incubation of MAO-B with the irreversible inhibitor pargyline at 10⁄IC50, and dilution of the enzyme–inhibitor complex to 0.1⁄IC50, the MAO-B activities are not fully recovered (less than 10% of control). 2.4. Kinetic study of hMAO-B inhibition Compound 6e was also used to further investigate the mode of MAO-B inhibition. The type of MAO-B inhibition was determined by Michaelis-Menten kinetic experiments [40]. The catalytic rates were measured at five different p-tyramine concentrations (50–3 000 lM), and each plot was constructed at four different concentrations of compound 6e (0, 0.5, 1.0 and 2.0 lM). The overlaid reciprocal Lineweaver-Burk plots (Fig. 3) showed that the plots for different concentrations of compound 6e were linear and intersected at the y-axis. This pattern indicated that compound 6e was a competitive MAO-B inhibitor, and these results further proved that the resveratrol-indazole hybrids were reversible MAO-B inhibitors.

Fig. 5. Inhibition of self-induced Ab (1–42) aggregation by compounds 6a–6e, 7, 8 and 9 comparing with that of curcumin. The thioflavin-T fluorescence method was used and the measurements were carried out in the presence of 20 lM test compound. The mean ± SD values from three independent experiments were shown.

stacking interaction (3.05 Å). However, for MAO-A, although compound 6e could enter the binding pocket of MAO-A, it didn’t show any interaction with MAO-A (Fig. 4A and C). In addition, the entrance cavity was a hydrophobic subpocket existing only in the MAO-B isoform. Therefore, such different binding modes of compound 6e in MAO-A/B might explain why compound 6e could selectively inhibit MAO-B.

2.6. Inhibition of self-induced Ab (1–42) aggregation All compounds tested for MAO inhibition were also valued through a thioflavin T-based fluorometric assay for their ability to inhibit self-induced Ab (1–42) aggregation [42,43]. Curcumin and resveratrol were used as references, because of its known activity in inhibition of Ab (1–42) self-aggregation. The results were gathered (Table 2 and Fig. 5), and it could be seen that compounds 6a–6f, 7, 8 and 9 exhibited good potencies (42.9–61.5% at 20 lM, IC50 = 18.8–23.6 lM) compared with curcumin (54.6% at 20 lM, IC50 = 19.8 lM) and resveratrol (64.2% at 20 lM, IC50 = 17. 9 lM). It was notable that the compounds 6e (58.9% at 20 lM, IC50 = 19.5 lM) and 9 (61.5% at 20 lM, IC50 = 18.8 lM) showed most effective. Compared with compound 6a, the introduction of 2-OH, compound 6b, reduced Ab (1 4 2) inhibitory activity, while compounds 6c–6f slightly increased the inhibitory activities on self-induced Ab (1–42) aggregation. Among compounds 7–9, compound 9 bearing a chromone revealed a good inhibition on selfinduced Ab (1 4 2) aggregation with inhibition, which was better than that of compound 7 and compound 8. From the results mentioned above, it can be easily concluded that the size of a substituent might crucially affect the Ab aggregation inhibition.

2.7. Docking study of compound 6e with Ab (1–42) peptide 2.5. Molecular modeling studies The binding mode of compound 6e with respect to MAO was investigated based on the X-ray crystal structures of MAO-B (PDB code 2V61) and MAO-A (PDB code 2Z5X). And the molecular docking study was performed using software package MOE 2008.10.23 [41]. The 3D and 2D pictures of binding were illustrated in Fig. 4. As Fig. 4B and D shown, compound 6e could occupy the entire binding pocket of MAO-B [18]. The indazole ring was hosted into the substrate cavity, which showed aromatic p-p stacking interactions with Tyr 435 and Tyr 398 (the ring-to-ring distances were 2.76 Å and 3.60 Å, respectively). The benzene ring occupied the entrance cavity, and it could also interact with Tyr 326 via p-p

To further study the interaction mode of compound 6e for Ab (1–42), molecular docking study was performed using software package MOE 2008.10 [41]. The X-ray crystal structure of the protein Ab structure (PDB 1IYT) from the Protein Data Bank was used in the docking study. As revealed in Fig. 6, the indazole ring interacted with the His 6 via cation–p stacking interactions with the distance of 2.63 Å. And the phenyl ring of compound 6e was an aromatic p-p stacking interaction with Tyr 10 with the ring-toring distance of 3.68 Å. These results indicated that the p-p and cation–p stacking interactions played important roles in the stability of the 6e/Ab (1–42) complex. Moreover, we have attached binding energy tables of Ab (1–42) in Fig. S2.

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Fig. 6. (A) 3D docking model of compound 6e with Ab (1–42). Atom colors: yellow-carbon atoms of 6e, gray-carbon atoms of residues of Ab, dark blue-nitrogen atoms, redoxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (B) 2D schematic diagram of docking model of compound 6e with Ab (1–42). The figure was prepared using the ligand interactions application in MOE.

2.8. In vitro blood–brain barrier permeation assay For successful central nervous system (CNS) drugs, the first requirement is crossing the blood–brain barrier (BBB) to reach brain. To evaluate the potential for these compounds to penetrate into brain, a parallel artificial membrane permeation assay for BBB (PAMPA-BBB) was used, which was described by Di et al. [44]. Assay validation was completed by comparing experimental permeabilities of 8 commercial drugs with reported values (Table 3). A plot of experimental data versus bibliographic values gave a good linear correlation, Pe (exp.) = 1.2014Pe (bibl.) – 0.3139 (R2 = 0.95). From this equation and taking into account the limit established by Di et al. for blood-brain barrier permeation, we classified compounds as follows: (a) ‘CNS +’ (high BBB permeation predicted): Pe (10 4.5. (b) ‘CNS ’ (low BBB permeation predicted): Pe (10 2.1.

6

cm s

1

6

cm s

1

Table 3 Permeability (Pe  10 6 cm s 1) in the PAMPA-BBB assay for 8 commercial drugs, used in the experiment validation.

a b

Commercial drugs

Bibla

PBS:EtOH (70:30)b

Testosterone Verapamil Beta-Estradiol Clonidine Corticosterone Hydrocortisone Lomefloxacin Ofloxacin

17 16 12 5.3 5.1 1.9 1.1 0.8

23.23 ± 1.05 18.16 ± 0.89 11.19 ± 0.82 4.15 ± 0.67 6.27 ± 0.72 3.12 ± 0.16 1.27 ± 0.13 1.32 ± 0.15

Taken from Ref. [44]. Data are the mean ± SD of three independent experiments.

)>

)<

(c) ‘ CNS +/ ’ (BBB permeation uncertain): Pe (10 from 4.5 to 2.1.

6

cm s

1

)

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Table 4 Permeability (Pe  10 6cm s 1) in the PAMPA-BBB assay for novel derivatives and their predictive penetration in the CNS. 6

Compound

Pe  10

6e 9

10.8 ± 0.8 4.9 ± 0.4

(cm s

1

)

4. Experimental section 4.1. Chemistry

Prediction CNS+ CNS+

All chemicals (reagent grade) used were purchased from Sino pharm Chemical Reagent Co., Ltd. (China). Reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qing-Dao, China) plates and the spots were detected under UV light (254 nm). Melting point was measured on an XT-4 micromelting point instrument and uncorrected. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer. 1H NMR and 13C NMR spectra were measured on a BRUKER AVANCE III spectrometer at 25 °C and referenced to TMS. Chemical shifts are reported in ppm (d) using the residual solvent line as internal standard. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. The purity of all compounds was confirmed to be higher than 95% through analytical HPLC performed with Agilent 1200 HPLC System. Mass spectra were obtained on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS) and Synapt G2 quadrupole time-of-flight (Q/TOF) tandem mass spectrometry (HRESI-MS). 4.2. General procedure for the preparation of compounds 6a–6e, 7, 8 and 9

Fig. 7. (A) Effects of compounds 6e and 9 on cell viability in PC12 cells. The cell viability was determined by the MTT assay after 24 h of incubation with various concentrations. The results were expressed as a percentage of control cells. Values are reported as the mean ± SD of three independent experiments. Data represent the mean SD of three observations.

Finally compounds 6e and 9 with good activities against Ab (1–42) aggregation and MAO were selected and the Pe values of are summarized in Table 4. It can be seen that compounds 6e and 9 might be able to penetrate the BBB. 2.9. Cells toxicity Based on the screening results above, compounds 6e and 9 with good MAO-B inhibition and significant inhibition of self-induced Ab aggregation, were selected to further examine the potential toxicity effect on the PC12 cells. After incubating the cells with compound 6e and 9 for 48 h, the cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. As shown in Fig. 7, the result revealed that compounds 6e and 9 at 6.25–100 lM did not have neurotoxicity. This suggested that compounds 6e and 9 might be used to develop promising drug candidates for the therapy of neurodegenerative diseases. 3. Conclusions In conclusion, a new series of resveratrol-indazole hybrids were designed, synthesized and evaluated as multitarget-directed antiAD agents for their inhibitory activities on MAO-B and Ab (1–42) aggregation. According to our screening results, compound 6e exhibited good MAO-B inhibition. Enzyme kinetics and molecular modeling studies revealed that the compound 6e could occupy the substrate binding site in hMAO-B. Compound 6e also showed inhibitory percentages similar to the reference compound resveratrol in the inhibition of Ab (1–42) self-aggregation assay. Besides, these compounds showed no toxicity to PC12 cells at 100 lM with brain penetration capacity for CNS activity. Altogether, the multifunctional effects of these resveratrol-indazole hybrids qualify them as potential MAO/Ab (1–42) aggregation inhibitors, and compound 6e might be a latent lead compound for further research.

Compounds 6a–6e, 7, 8 and 9 were easily prepared as described in the literature with some modification [35]. In brief, the mixture of aromatic aldehyde and aniline were stirred in a small amount of ethanol at reflux condition for about 4–12 h then followed by filtration and recrystallization in EtOH or MeOH to obtain the pure compounds. 4.2.1. (E)-N-benzylidene-1H-indol-5-amine (6a) Yield 85%; gray solid; IR mmax/cm 1 (KBr) 3108.60, 1627.98, 1501.56, 1448.86, 1290.27, 1074.11, 956.94, 887.50, 810.76, 743.80, 690.28; m.p. 197.1–197.9 °C; ESI/MS m/z: 222.1 [M+H]+; 1 H NMR (400 MHz, DMSO d6) d 13.13 (s, 1H), 8.74 (s, 1H), 8.10 (s, 1H), 7.97 (dd, J = 6.4, 3.0 Hz, 2H), 7.66 (d, J = 1.0 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.56–7.49 (m, 3H), 7.43 (dd, J = 8.8, 1.8 Hz, 1H). 13 C NMR (100 MHz, DMSO d6) d 159.5, 145.1, 139.2, 136.8, 134.4, 131.6, 129.3, 129.3, 128.9, 128.9, 123.9, 121.9, 111.9, 111.1. HRMS: calcd for C14H11N3 [M+H]+ 222.1031, found 222.1036. 4.2.2. (E)-2-(((1H-indazol-5-yl)imino)methyl)phenol (6b) Yield 83%; yellow solid; IR mmax/cm 1 (KBr) 3137.77, 1629.33, 1505.41, 1456.47, 1273.30, 954.96, 801.59, 668.70; m.p. 217.9–2 18.3 °C; ESI/MS m/z: 238.0 [M+H]+; 1H NMR (400 MHz, DMSO d6) d 13.37 (s, 1H), 13.21 (s, 1H), 9.06 (s, 1H), 8.14 (s, 1H), 7.83 (s, 1H), 7.56 (m, 4H), 6.99 (s, 2H). 13C NMR (100 MHz, DMSO d6) d 162.4, 160.7, 141.7, 139.5, 134.6, 133.3, 132.9, 123.8, 121.4, 119.9, 119.6, 117.0, 113.0, 111.5. HRMS: calcd for C14H13N3O [M+H]+ 238.0980, found 238.0982. 4.2.3. (E)-3-(((1H-indazol-5-yl)imino)methyl)phenol (6c) Yield 85%; gray solid; IR mmax/cm 1 (KBr) 3126.35, 1627.53, 1576.06, 1506.54, 1473.07, 1274.39, 951.98, 874.57, 684.52; m.p. 179.1–182.6 °C; ESI/MS m/z: 238.0 [M+H]+; 1H NMR (400 MHz, DMSO d6) d 13.12 (s, 1H), 9.71 (s, 1H), 8.64 (s, 1H), 8.10 (s, 1H), 7.64 (s, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.36 (m, 4H), 6.93 (d, J = 7.0 Hz, 1H). 13C NMR (100 MHz, DMSO d6) d 159.5, 158.2, 145.1, 139.2, 138.2, 134.4, 130.3, 123.9, 121.9, 120.6, 118.9, 114.6, 111.8, 111.1. HRMS: calcd for C14H13N3O [M+H]+ 238.0980, found 238.0978.

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4.2.4. (E)-N-(3,4-dimethoxybenzylidene)-1H-indazol-5-amine (6d) Yield 86%; gray solid; IR mmax/cm 1 (KBr) 3150.74, 1633.79, 1584.17, 1507.46, 1457.90, 1228.07, 1133.64, 940.91, 882.67, 674.34; m.p. 170.0–174.8 °C; ESI/MS m/z: 282.2 [M+H]+; 1H NMR (400 MHz, DMSO d6) d 13.10 (s, 1H), 8.61 (s, 1H), 8.08 (s, 1H), 7.63–7.51 (m, 3H), 7.46 (d, J = 8.2 Hz, 1H), 7.42–7.36 (m, 1H), 7.10 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO d6) 159.1, 152.0, 149.5, 145.4, 139.0, 134.3, 129.8, 124.2, 123.9, 122.0, 111.8, 111.4, 111.0, 109.8, 56.1, 55.9. HRMS: calcd for C16H15N3O2 [M+H]+ 282.1243, found 282.1248. 4.2.5. (E)-N-(3,4-dichlorobenzylidene)-1H-indazol-5-amine (6e) Yield 90%; gray solid; IR mmax/cm 1 (KBr) 3180.90, 1635.68, 1548.39, 1502.25, 1470.17, 1124.55, 1026.75, 952.40, 886.02, 791.54, 696.79; m.p. 221.4–225.2 °C; ESI/MS m/z: 290.1 [M+H]+; 1 H NMR (400 MHz, DMSO d6) d 13.17 (s, 1H), 8.77 (s, 1H), 8.15 (d, J = 19.8 Hz, 2H), 7.93 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7.60 (d, J = 6.9 Hz, 1H), 7.47 (s, 1H). 13C NMR (100 MHz, DMSO d6) 161.6, 149.0, 144.2, 142.2, 139.4, 138.6, 137.0, 136.4, 135.1, 133.4, 128.6, 126.4, 117.5, 116.0. HRMS: calcd for C14H9N3Cl2 [M+H]+ 290.0252, found 290.0246. 4.2.6. (E)-4-(((1H-indazol-5-yl)imino)methyl)-2-methoxyphenol (6f) Yield 84%; yellow solid; IR mmax/cm 1 (KBr) 3160.45, 1630.39, 1576.27, 1505.19, 1462.75, 1221.81, 946.28, 883.52, 686.19; m.p. 131.4–135.1 °C; ESI/MS m/z: 268.2 [M+H]+; 1H NMR (400 MHz, DMSO d6) d 13.08 (s, 1H), 8.55 (s, 1H), 8.07 (s, 1H), 7.62–7.49 (m, 3H), 7.45–7.29 (m, 3H), 6.91 (d, J = 8.1 Hz, 1H), 3.86 (s, 3H). 13 C NMR (100 MHz, DMSO d6) d 159.3, 150.4, 148.5, 145.6, 139.0, 134.3, 128.6, 126.5, 124.3, 122.0, 115.9, 111.2, 111.0, 110.8, 56.0. HRMS: calcd for C14H13N3O2 [M + H]+ 268.1086, found 268.1088.

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4.3. In vitro inhibition of monoamine oxidase [36] Human MAO-A and MAO-B were purchased from SigmaAldrich. The capacity of the test compounds to inhibit MAO-A and MAO-B activities was assessed by Amplex Red MAO assay. Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing the test drugs at various concentrations and adequate amounts of recombinant hMAO-A or hMAO-B required and adjusted to obtain in our experimental conditions the same reaction velocity, i.e., to oxidize (in the control group) the same concentration of substrate: 165 pmol of p-tyramine/min (hMAO-A: 1.1 lg protein; specific activity: 150 nmol of p-tyramine oxidized to p-h ydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 lg protein; specific activity: 22 nmol of p-tyramine transformed/min/ mg protein) were incubated for 15 min at 37 °C in a flat-blackbottom 96-well microtest plate placed in a dark fluorimeter chamber. After this incubation period, the reaction was started by adding 200 lM (final concentrations) Amplex Red reagent, 1 U/mL horseradish peroxidase, and 1 mM p-tyramine. The production of H2O2 and consequently, of resorufin, was quantified at 37 °C in a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA) mutimode detection platform reader based on the fluorescence generated (excitation, 545 nm; emission, 590 nm). The specific fluorescence emission was calculated after subtraction of the background activity. The background activity was determined from wells containing all components except the hMAO isoforms, which were replaced by a sodium phosphate buffer solution (0.05 M, pH 7.4). The percent inhibition was calculated by the following expression: (1d IFi/IFc)  100 in which IFi and IFc are the fluorescence intensities obtained for hMAO in the presence and absence of inhibitors after subtracting the respective background. 4.4. Reversibility and irreversibility study [37]

4.2.7. (E)-N-(pyridin-4-ylmethylene)-1H-indazol-5-amine (7) Yield 85%; gray solid; IR mmax/cm 1 (KBr) 3152.46, 1600.26, 1486.00, 1418.08, 1213.46, 1138.46, 932.75, 892.55, 802.18, 664.17; m.p. > 250 °C; ESI/MS m/z: 223.1 [M+H]+; 1H NMR (400 MHz, DMSO d6) d 13.20 (s, 1H), 8.82 (s, 1H), 8.76 (d, J = 4.3 Hz, 2H), 8.14 (s, 1H), 7.89 (d, J = 5.0 Hz, 2H), 7.77 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO d6) d 162.3, 155.6, 155.6, 148.8, 148.1, 144.4, 139.5, 128.6, 127.3, 127.3, 126.4, 118.0, 116.0. HRMS: calcd for C13H10N4 [M+H]+ 223.0984, found 223.0979. 4.2.8. (E)-N-(furan-2-ylmethylene)-1H-indazol-5-amine (8) Yield 86%; yellow solid; IR mmax/cm 1 (KBr) 3107.69, 1624.53, 1496.31, 1472.87, 1206.26, 1143.96, 930.19, 881.24, 801.91, 744.06, 668.55; m.p. 191.4–194.0 °C; ESI/MS m/z: 212.1 [M+H]+; 1 H NMR (400 MHz, DMSO d6) d 13.12 (s, 1H), 8.55 (s, 1H), 8.09 (s, 1H), 7.95 (s, 1H), 7.64 (s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 3.3 Hz, 1H), 6.72 (d, J = 1.6 Hz, 1H). 13C NMR (100 MHz, DMSO d6) d 152.6, 147.3, 146.5, 144.8, 139.2, 134.5, 123.9, 121.6, 116.6, 112.9, 112.0, 111.1. HRMS: calcd for C12H9N3O [M+H]+ 212.0824, found 212.0822. 4.2.9. (E)-3-(((1H-indazol-5-yl)imino)methyl)-4H-chromen-4-one (9) Yield 88%; yellow solid; IR mmax/cm 1 (KBr) 3168.00, 1647.51, 1460.33, 1277.87, 1205.11, 968.91, 873.88, 809.70, 754.40; m.p. 1 81.4–183.6 °C; ESI/MS m/z: 290.1 [M+H]+; 1H NMR (400 MHz, DMSO d6) d 13.15 (s, 1H), 8.16 (d, J = 12.7 Hz, 1H), 8.07 (s, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.76 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.52 (t, J = 6.9 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.14 (t, J = 7.3 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 5.97 (s, 1H). 13C NMR (100 MHz, DMSO d6) d 179.9, 155.9, 146.8, 138.0, 134.6, 133.8, 133.7, 126.0, 123.7, 123.2, 122.2, 118.6, 118.4, 111.9, 107.2, 103.3, 100.6. HRMS: calcd for C17H11N3O2 [M+H]+ 290.0930, found 290.0928.

The reversibility of MAO-B inhibition was determined by dilution assay. At concentrations equal to 10⁄IC50 and 100⁄IC50 for hMAO-B inhibition, compound 5 m was incubated with the enzyme (0.75 mg/ml) for 30 min at 37 °C in PBS (0.05 M, pH 7.4). The parallel control was conducted by replacing the compound with buffer, and the corresponding amount of DMSO was added as co-solvent to all incubations. After the incubation period, the complex was diluted 100-fold to obtain final concentrations of compound 6e equal to 0.1 ⁄ IC50 and 1 ⁄ IC50. For comparison, pargyline were incubated with hMAO-B at concentrations of 10 ⁄ IC50 in similar manner and diluted to 0.1 ⁄ IC50. The residual enzyme catalytic rates were determined following the method for the IC50 determination and all results were expressed as mean ± SD. 4.5. Kinetic study of MAO-B inhibition [38] To obtain of the mechanism of action 6e, reciprocal plots of 1/velocity versus 1/substrate were constructed at different concentrations of the substrate p-tyramine (50–3000 lM). Four different concentrations of 6e (0, 0.75, 1.50 and 300 lM) were selected for the kinetic analysis of MAO-B inhibition. The plots were assessed by a weighted least-squares analysis that assumed the variance of velocity (v) to be a constant percentage of v for the entire data set. Slopes of these reciprocal plots were then plotted against the concentration of 6e in a weighted analysis. Data analysis was performed with Graph Pad Prism 4.03 software (Graph Pad Software Inc.). 4.6. Inhibition of Ab (1-42) self-induced aggregation [40,41] Inhibition of self-induced Ab (1–42) aggregation was measured using a Thioflavin T (ThT)-binding assay. HFIP pretreated Ab (1–42)

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samples (Anaspec Inc) were resolubilized with a 50 mM phosphate buffer (pH 7.4) to give a 25 lM solution. Each tested compound was firstly prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and 1 lL of each was added to the well of black, opaque Corning 96-well plates such that the final solvent concentration was 10%. The final concentration of each compound was prepared in independent triplicates. The solvent control was also included. Then, 9 lL of 25 mM Ab (1–42) sample was added to each well and the samples mixed by gentle trapping. Plates were covered to minimize evaporation and incubated in dark at room temperature for 46–48 h with no agitation. After the incubation period, 200 lL of 5 lM ThT in 50 mM glycine-NaOH buffer (pH 8.0) was added to each well. Fluorescence was measured on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA) multi-mode plate reader with excitation and emission wavelengths at 446 nm and 490 nm, respectively. The fluorescence intensities were compared and the percent inhibition due to the presence of the inhibitor was calculated by the following formula: 100 (IFi/IFo  100) where IFi and IFo are the fluorescence intensities obtained for Ab (1–42) in the presence and in the absence of inhibitor, respectively. 4.7. Molecular modeling studies [39] Molecular modeling calculations and docking studies were performed using Molecular Operating Environment (MOE) software version 2008.10 (Chemical Computing Group, Montreal, Canada). The X-ray crystallographic structures of hMAO-A (PDB code 2Z5X), hMAO-B (PDB code 2 V61) and Ab (1–42) (PDB code PDB 1IYT) were obtained from the Protein Data Bank. All water molecules in PDB files were removed and hydrogen atoms were subsequently added to the protein. The compound 6e was built using the builder interface of the MOE program and energy minimized using MMFF94x force field. Then the 6e was docked into the active site of the protein by the ‘‘Triangle Matcher” method, which generated poses by aligning the ligand triplet of atoms with the triplet of alpha spheres in cavities of tight atomic packing. The Dock scoring in MOE software was done using ASE scoring function and Force field was selected as the refinement method. The best 10 poses of molecules were retained and scored. After docking, the geometry of resulting complex was studied using the MOE’s pose viewer utility.

standards of known BBB permeability were included to validate the analysis set. 4.9. Cell culture and measurement of cell viability The toxicity effect of the tested compounds on the rat pheochromocytoma (PC12) cells was examined according to previously reported methods. PC 12 cells was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and routinely grown at 37 °C in a humidified incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum, 100 units per mL penicillin, and 100 units per mL of streptomycin. Cells were sub-cultured in 96-well plates at a seeding density of 5000 cells per well and allowed to adhere and grow. When cells reached the required confluence, they were placed into serum-free medium and treated with compounds 6e and compounds 9. Twenty-four hours later the survival of cells was determined by MTT assay. Briefly, after incubation with 20 lL of MTT at 37 °C for 4 h, living cells containing MTT formazan crystals were solubilized in 200 lL DMSO. The absorbance of each well was measured using a microculture plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm. Results are expressed as the mean ± SD of three independent experiments. Acknowledgments This work was supported by the programs of the National Natural Science Foundation of China [grant numbers 81403175 and 81274200]; Project of the Shanghai Committee of Science and Technology [grant number 13401900301]; Project of the Shanghai Municipal Commission of Health and Family planning [grant number 2017YQ072 and 201740152]; Research Fund for the Doctoral Program of Shanghai [grant number B201703]; Research Fund of Jiangxi University of Traditional Chinese Medicine [Grant No. 2015BS008]; Project from Health and Family planning Commission of Jiangxi province [Grant No. 20173013] and Xinglin Scholar Plan. Conflict of Interest The authors declare no conflict of interest. Appendix A. Supplementary material

4.8. In vitro blood–brain barrier permeation assay [42] Brain penetration of compounds was evaluated using a parallel artificial membrane permeation assay (PAMPA) in a similar manner as described by Di et al. Commercial drugs were purchased from Sigma and Alfa Aesar. The porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and the acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR@) was from Corning Incorporated. The acceptor 96-well microplate was filled with 300 lL of PBS/EtOH (7:3), and the filter membrane was impregnated with 4 lL of PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to achieve a concentration of 100 mg/mL, 200 lL of which was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 16 h at 25 °C. After incubation, the donor plate was carefully removed and the concentration of compound in the acceptor wells was determined using an UV plate reader (Flexsta-tion@3). Every sample was analyzed at five wavelengths, in four wells, in at least three independent runs, and the results are given as the mean ± SD. In each experiment, 8 quality control

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