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. 2024 May 20;15(6):798–805. doi: 10.1021/acsmedchemlett.3c00532

Benzodioxane Carboxamide Derivatives As Novel Monoamine Oxidase B Inhibitors with Antineuroinflammatory Activity

Demeng Sun 1, Bo Wang 1, Yanmei Jiang 1, Zuo Kong 1, Mengxue Mu 1, Changhuan Yang 1, Jingbo Tan 1, Yun Hu 1,*
PMCID: PMC11181489  PMID: 38894921

Abstract

graphic file with name ml3c00532_0009.jpg

In this study, a series of N-phenyl-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide derivatives were designed, synthesized, and evaluated for their inhibitory activities against human MAO-B (hMAO-B). The structure–activity relationship (SAR) was investigated and summarized. Compound 1l (N-(3,4-dichlorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide) showed the most potent inhibitory activity with an IC50 value of 0.0083 μM and the selectivity index (IC50 (hMAO-A)/IC50 (hMAO-B)) was >4819. Kinetics and reversibility studies confirmed that compound 1l acted as a competitive and reversible inhibitor of hMAO-B. Molecular docking studies revealed the enzyme–inhibitor interactions, and the rationale was provided. Additionally, compound 1l could effectively inhibit the release of NO, TNF-α, and IL-1β in both LPS- and Aβ1–42-stimulated BV2 cells and attenuate the cytotoxicity induced by Aβ1–42. Since compound 1l exhibited low neurotoxicity, we believe that the hit compound with dual activities of inhibiting MAO-B and antineuroinflammation could be further investigated as a novel potential lead for future studies in vivo.

Keywords: monoamine oxidase B; reversible inhibitors; antineuroinflammation; 1,4-benzodioxan

Monoamine oxidases (MAOs) are important flavin-dependent enzymes in the metabolism of monoamine neurotransmitters, which consist of two isoforms: MAO-A and MAO-B. In the human body, MAO-A predominates in sympathetic nerve terminals and intestinal mucosa, whereas MAO-B is the predominant isoform expressed in the brain.1 MAOs catalyze the oxidative deamination of monoamines, such as dopamine and tyramine, resulting in the production of aldehydes and hydrogen peroxide (H2O2). In the presence of trace amounts of metals, H2O2 can be converted to highly neurotoxic hydroxyl radicals, which would increase the oxidative stress.2 Evidence show that the expression and activity of MAO-B significantly increase in the brain of patients with Parkinson’s disease (PD) and Alzheimer’s disease (AD), suggesting that MAO-B could contribute to neurodegenerative processes by reducing the levels of neurotransmitters and increasing the oxidative stress.3,4

MAO-B inhibition is expected to increase the availability of neurotransmitters and reduce the oxidative stress in the brains of patients. Thus, MAO-B inhibitors are considered to be promising therapeutic agents for PD and AD.5 Several MAO-B inhibitors have been prescribed in the clinical treatment of PD, or assessed in the clinical trial of AD patients, such as R-(−)-deprenyl, rasagiline, and safinamide (Figure. 1A).6−9 The first two belong to irreversible inhibitors, and the latter is a reversible inhibitor. Reversible MAO-B inhibitors are deemed to have safer profiles, because of their noncovalent interaction with the enzyme, which could overcome the shortcomings of the irreversible inhibitors such as causing permanent damage to the enzyme and potential immunogenicity of enzyme–inhibitor adducts.10 Hence, more efforts will be devoted to the development of new reversible inhibitors of MAO-B in the future.

Figure 1.

Figure 1

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(A) Structures of known irreversible and reversible MAO inhibitors. (B) Structures and potencies of previously described MAO-B inhibitors and the design strategy of 1,4-benzodioxan-6-carboxamide derivatives.

Neuroinflammation is another commonality shared in neurodegenerative diseases, which is marked by the production of pro-inflammatory mediators, including NO, TNF-α, and IL-1β in the central nervous system.11 The increasing release of pro-inflammatory mediators can lead to synaptic dysfunction, neuronal death, and inhibition of neurogenesis.12 One of the main source of pro-inflammatory mediators is the reactive microglia that are activated by pathological stimuli, including lipopolysaccharide (LPS) and amyloid β (Aβ).13,14 Suppressing the microglial activation and reducing the level of pro-inflammatory mediators has been recognized as an attractive strategy for the treatment of neuroinflammation-related diseases.15

In the search for novel MAO-B inhibitors, several lines of evidence have shown that 1,4-benzodioxan (2,3-dihydrobenzo[b][1,4]dioxine) is a privileged structure for the rational design of lead compounds. Engelbrecht et al.16 synthesized a series of 6-benzyloxybenzodioxane derivatives and found that the most potent compound (7c) exhibited an inhibitory activity with an IC50 value of 0.045 μM. We had also investigated and reported the 1,4-benzodioxan-substituted chalcone derivatives as potent selective and reversible MAO-B inhibitors.17 The representative compound 22 exhibited an IC50 value of 0.026 μM with a selectivity index value of at least >1538. Interestingly, 1,4-benzodioxan derivatives have been also found to demonstrate anti-inflammatory activities that could effectively reduce the expression of pro-inflammatory mediator in LPS-stimulated macrophages.18−20

Inspired by these results, we hypothesize that a novel series of 1,4-benzodioxan derivatives combining potent MAO-B inhibiting and antineuroinflammation activities are feasible, which would exhibit improved therapeutic benefits for the management of PD and AD.

Evidence shows that the 1,4-benzodioxan group is a useful scaffold for the design of new MAO-B inhibitors and substitution on the C6 position of 1,4-benzodioxan ring could yield potent selective MAO-B inhibitors.16,17 Guided by the structure–activity relationship (SAR) results in the previous studies, a series of N-phenyl-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide derivatives were designed in the present work. The rational design strategy was mainly pursued as follows (Figure 1B):

  • (1)

    the 1,4-benzodioxan ring was retained as a pharmacophore of the designed compounds;

  • (2)

    on the C6 position of the 1,4-benzodioxan ring, the carboxamide moiety was inserted as a linker; and

  • (3)

    the amino group of the linker was substituted using a phenyl ring with various substituents of different type, number, and position.

As a result, a library of N-phenyl-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide derivatives were successfully obtained by amide coupling reactions, according to the synthetic protocol outlined in Scheme 1.

Scheme 1. Synthesis of Compounds 1a–1x, 2a–2c, 3a, 3b, and 4.

Scheme 1

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All the synthetic compounds were first screened for the inhibitory effects on human MAO-B and MAO-A using a fluorescence-based assay. The inhibition rate (% of control) was tested at 1 μM, unless otherwise stated. The irreversible inhibitor rasagiline and reversible inhibitor safinamide were used as positive controls for hMAO-B inhibition. Clorgyline was used as a positive control for hMAO-A inhibition. As shown in Table 1, rasagiline and safinamide shown significant inhibitory activities on hMAO-B. The positive controls exhibited 90.1% and 86.9% inhibition at 1 μM with IC50 values of 0.096 μM and 0.060 μM, respectively, which are in accordance with that of previous reports.21,22 The parent compound N-phenyl-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide 1a exhibited a moderate inhibition effect against hMAO-B (16.1% inhibition) with an IC50 of 7.66 μM. However, replacing the N-phenyl of 1a with the benzyl moiety (1b) resulted in a marked decreased inhibition (<5%), which suggested that a length of two atoms of the amide linker is necessary for the inhibitory activity. When replacing the 1,4-benzodioxan moiety of 1a with the phenyl (2a) or 1,3-dioxaindane (1c) moiety, both resulted compounds showed little inhibition (<5%) indicating that the 1,4-benzodioxan moiety is an essential pharmacophore for hMAO-B inhibition. As for the compound 1a, introducing −F to the ortho position of N-phenyl (1d) led to a comparable inhibition (19.1% inhibition) while −Cl was not tolerated at the same position (1e, <5% inhibition). At the meta position of the N-phenyl, introducing – F, – Cl and – Br led to 6-fold to 98-fold higher hMAO-B inhibition compared to 1a. The resulting compounds 1f–1h exhibited IC50 values of 1.26,, 0.07, and 0.17 μM, respectively. At the para position, introducing −F, −Cl, and −Br also led to increased inhibition (1i–1k, 41.8%, 71.1%, and 51.4% inhibition, respectively). The 3–F-, 3–Cl-, and 3–Br-substituted compounds (1i–1k) exhibited IC50 values of 1.21, 0.33, and 1.18 μM, repsectively, which were ∼6-fold and 23-fold more potent than 1a. These results suggested that the introduction of halogen atoms at the meta and para positions of the N-phenyl of 1a were favorable to increase the inhibitory potency and the −Cl group would be preferable.

Table 1. hMAO Inhibitory Activities of Compounds 1a–1x, 2a–2c, 3a, 3b, and 4a.

  hMAO-A hMAO-B
  
compound IC50 (μM) inhibition (%) IC50 (μM) selectivity index, SIb
1a >40 16.1 ± 2.9 7.66 ± 1.2 >5
1b >40 <5 N.D. N.D.
1c >40 <5 N.D. N.D.
1d >40 19.1 ± 0.77 N.D. N.D.
1e >40 <5 N.D. N.D.
1f >40 61.3 ± 0.025 1.26 ± 0.23 >31
1g >40 84.9 ± 1.85 0.07 ± 0.014 >571
1h >40 75.5 ± 2.8 0.17 ± 0.02 >235
1i >40 41.8 ± 7.1 1.21 ± 0.16 >33
1j >40 71.1 ± 2.58 0.33 ± 0.06 >121
1k >40 51.4 ± 4.5 1.18 ± 0.36 N.D.
1l >40 91.4 ± 0.74 0.0083 ± 0.002 >4819
1m >40 81.2 ± 6.6 0.021 ± 0.004 >1904
1n >40 78.1 ± 2.3 0.12 ± 0.04 >333
1o >40 82.1 ± 7.6 0.049 ± 0.02 >816
1p >40 84.5 ± 0.6 0.058 ± 0.003 >689
1q >40 85.3 ± 0.96 0.038 ± 0.0007 >1052
1r >40 53.0 ± 6.8 N.D. N.D.
1s >40 19.7 ± 0.34 N.D. N.D.
1t >40 <5 N.D. N.D.
1u >40 <5 N.D. N.D.
1v >40 65.8 ± 0.26 0.45 ± 0.01 >88
1w >40 27.4 ± 1.8 N.D. N.D.
1x >40 86.8 ± 0.18 0.036 ± 0.01 >1111
2a >40 <5 N.D. N.D.
2b >40 <5 N.D. N.D.
2c >40 40.2 ± 1.02 N.D. N.D.
3a >40 13.8 ± 12.9 N.D. N.D.
3b >40 36.1 ± 0.04 N.D. N.D.
4 >40 30.8 ± 1.7 N.D. N.D.
rasagiline N.D. 90.1 ± 0.9 0.096 ± 0.01 1067d
safinamide N.D. 86.9 ± 0.7 0.060 ± 0.001 4335d
clorgyline 79.9% ± 0.46%c N.D. N.D. N.D.
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a

The inhibition rate (%) was calculated at the concentration of 1 μM of tested compounds, unless otherwise stated. The data are represented as mean ± SD from at least three independent experiments. N.D. = not determined.

b

SI = IC50(hMAO-A)/IC50(hMAO-B).

c

Percent inhibition at 0.1 μM.

d

Value obtained from ref (21).

Compounds bearing more halogen atoms at the N-phenyl position of 1a were then derivatized. The resulting compounds (1l–1q) substituted with two halogen atoms exhibited significant increased hMAO-B inhibition, compared to 1a. The most potent compound was the 3,4-dichlorine-substituted compound 1l with an IC50 value of 0.0083 μM, which was ∼11-fold more potent than rasagiline and 7-fold more potent than safinamide. The 3–Br, 4–Cl substituted compound 1m exhibited a relatively less potent inhibitory activity than 1l, the IC50 value of which was 0.021 μM. IC50 values of other compounds with the substitutions of – F, −Cl, and −Br at both the meta and para positions of N-phenyl (1n, 1o, 1p, and 1q) were 0.12, 0.049, 0.058, and 0.038 μM, respectively. Nevertheless, compound 1r with the substitutions of 2,3-dichlorine showed a marked decrease in inhibitory potency, compared to 1a. Only 53.0% hMAO-B inhibition was observed, which could be explained by the intolerance of the substitution of −Cl at the ortho position of N-phenyl, as described above.

The introduction of electron-donating groups such as −OCH3 to the N-phenyl of 1a was also investigated. Introducing the −OCH3 group to the meta position of N-phenyl (1s) led to a slightly higher inhibition (19.7% inhibition), compared to C31, while it was not tolerated at the para position (1t, <5% inhibition). The 3,4-dimethoxyl-substituted compound 1u also showed little hMAO-B inhibition (<5% inhibition). Considering that replacing the 3–Cl or 4–Cl of 1l with the −OCH3 group also resulted in markedly decreased inhibitory activities (1v, IC50 = 0.45 μM; 1w, 27.4% inhibition), we concluded that the introduction of electron-donating groups such as the −OCH3 to the N-phenyl was unfavorable to increase the inhibitory potency of these compounds.

To further explore the SAR of this series of compounds, additional reference compounds were prepared and investigated.

  • (1)

    As for compound 1a, replacing the N-phenyl of 1a with the indazole moiety (3a) led to a slightly lower inhibition (13.8% inhibition). While the compound with the replacement of 1,4-benzodioxan moiety (3b) at the same position showed 2.2-fold higher inhibition (36.1% inhibition), compared to 1a, indicating that the introduction of certain fused aromatic rings with proper volume might be favorable.

  • (2)

    As for the active compounds 1g and 1l, replacing the 1,4-benzodioxan moiety with the phenyl ring led to compounds 2b and 2c, both of which showed markedly decreased hMAO-B inhibition, compared to the corresponding compounds (<5% and 40.2% inhibition). Besides, replacing the 1,4-benzodioxan moiety of 1l with the 1,3-dioxaindane ring (1x) also resulted in a 4.3-fold decrease in inhibitory potency (IC50 = 0.036 μM). The above results reinforce that the 1,4-benzodioxan moiety is crucial for the inhibiting activity of this class of inhibitors.

  • (3)

    The importance of the spatial position of the amide group was explored finally. An exchange on the carbonyl and nitrogen group positions was performed for the most potent compound 1l. The resulting compound 4 exhibited a noticeable decrease on the inhibiting activity (30.8% inhibition), compared to 1l, which indicated that the carbonyl connecting with the C6 position of the 1,4-benzodioxan ring is also a very important structural requirement for the inhibitory potency.

Taking together, an overview of the SAR of the N-phenyl-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide derivatives, with respect to hMAO-B inhibition, is provided in Figure 2.

Figure 2.

Figure 2

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SAR of 1,4-benzodioxan carboxamide derivatives toward hMAO-B inhibition.

As for hMAO-A, all of the tested compounds showed <50% inhibition, even at 40 μM (Table 1). According to the definition of selectivity index (SI), which is defined as SI = IC50(hMAO-A)/IC50(hMAO-B), the most potent hMAO-B inhibitor 1l exhibited an SI value of >4819, which was superior to that of rasagiline (SI = 1067) and safinamide (SI = 4335).23 Thus, we concluded that the potent inhibitors found in this work are highly selective hMAO-B inhibitors.

To investigate the inhibiting mode of the active compounds, the most potent compound 1l was selected as a representative hMAO-B inhibitor in this work and subjected to an enzyme kinetics study. Based on the kinetics study, a Lineweaver–Burk graph was constructed in the absence or presence of inhibitors at various concentrations. As illustrated in Figure. 3A, the graphs of compound 1l in different concentrations were linear and intersect at the y-axis, which suggested that the active compound 1l behaved as a competitive hMAO-B inhibitor.

Figure 3.

Figure 3

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(A) Lineweaver–Burk plot for hMAO-B inhibition by compound 1l. (B) Time-dependent inhibition of hMAO-B by reference compounds rasagiline and safinamide (0.20 and 0.06 μM, respectively) and the tested compound 1l. The residual activity was expressed as a percentage of the control. (C) Dialysis study of MAO-B inhibition performed by reference compounds R-(−)-deprenyl, safinamide, and the tested compound 1l. The residual activity of undialysis and dialysis groups was expressed as a percentage of the control. Data were shown as mean ± SD of three independent experiments.

We next performed the time-dependent inhibition assay and dialysis assay to investigate the inhibitory reversibility of compound 1l. For the time-dependent inhibition study, the irreversible inhibitor rasagiline and the reversible inhibitor safinamide were used as reference compounds. After incubation with the inhibitors for 15 min, the enzyme was then added with the substrate benzylamine. The enzymatic activity (% of control) was measured for 120 min. As illustrated in Figure 3B, the activity of hMAO-B decayed continuously throughout the incubation period when treated with rasagiline, which indicated that the inhibition was irreversible.23 In the case of safinamide, when incubated with benzylamine, the enzymatic activity began to increase gradually after 48 min, indicating that the inhibitor can be replaced by the substrate, and the enzymatic activity could be recovered. Similar changes of enzymatic activity could be observed when treating the enzyme with 1l. The activity of hMAO-B began to recover 40 min after adding the substrate, which suggested that the inhibition of compound 1l on hMAO-B was reversible.

The results of the dialysis assay further confirmed the reversible inhibition of compound 1l. In the dialysis assay, MAO-B was incubated with the tested inhibitors for 15 min and subsequently dialyzed for 24 h. As shown in Figure 3C, after dialysis, the enzymatic activity of the irreversible inhibitor R-(−)-deprenyl-treated group was not recovered with the residual activity of 16.6% of the control, which was comparable to that of the undialyzed group. While the enzymatic activity of the safinamide-treated group was recovered from 46% to 90% after dialysis. As for the compound 1l, after dialysis, the enzymatic activity was recovered from 42.8% to 103% of the control, which distinctly showed that compound 1l was a reversible hMAO-B inhibitor.

To investigate the possible interaction mechanism between the active compounds and hMAO-B, we performed a molecular docking study using the docking module in the software MOE. The parent 1,4-benzodioxan carboxamide compound 1a and the most potent compound 1l were selected and docked into the active site of the cocrystal structure of hMAO-B (PDB code: 2V5Z). The hMAO-B active site consists of two cavities: the substrate cavity in front of the FAD on one side, and the entrance cavity located underneath the protein surface on the opposite side, which can be closed by the loop formed by residues 99–112.22 As shown in Figure 4A, compound 1a adopted an extended pose in the active site of hMAO-B. The N-phenyl ring occupied the hydrophobic pocket in the entrance cavity formed by Pro102, Pro104, Trp119, Phe168, Ile199, etc. On the other side, the 1,4-benzodioxan moiety was located at the substrate cavity formed by Ile198, Gln206, Phe343, Tyr398, Tyr435, etc. The 1,4-dioxane ring showed C–H···π interactions with Tyr398 and FAD (Figure S1). Besides, the carboxamide linker of 1a established a hydrogen bond with Cys172, of which the carboxylic oxygen served as a hydrogen bond acceptor. Compound 1l adopted a similar docking pose in the active site of hMAO-B (Figure 4A). The carboxylic oxygen of the carboxamide linker formed a hydrogen bond with Cys172. On the one hand side, the 1,4-benzodioxan moiety occupied the substrate cavity and showed C–H···π interactions with Tyr398 and FAD. On the opposite side, the 3,4-dichlorophenyl ring occupied the entrance cavity, and the phenyl ring formed a C–H···π interaction with Ile199 (Figure 4B). Furthermore, the 3,4-substituted chlorine atoms were embedded deeply in the highly lipophilic so-call halogen binding pocket positioned in the entrance cavity,24 which might reinforce the binding with the enzyme by enhancing the hydrophobic interactions and, consequently, lead to increased inhibitory activity.

Figure 4.

Figure 4

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(A) Docking poses of 1,4-benzodioxan carboxamide derivatives 1a and 1l in the active site of hMAO-B (PDB code: 2V5Z). (B) 2D diagram of compound 1l and its interactions with proximal residues in the active site of hMAO-B. Hydrogen bonds are shown as orange dotted lines. For the sake of clarity, only the relevant residue side chains are shown. FAD is rendered as white sticks. Ligands are rendered as sticks as follows: cyan, compound 1a; yellow, compound 1l.

We also attempted to explain why the active compounds exhibited weak inhibitory activity against hMAO-A by using a docking study. The most active compound 1l that showed the highest SI was selected as a case study in this series and docked into the active site of hMAO-A. It was found that the binding free energy of 1l with hMAO-A was much higher than that with hMAO-B (−5.7764 kcal/mol versus −8.7803 kcal/mol, Table S1), which indicated that 1l had a lower binding affinity with hMAO-A. As illustrated in Figure 5, in contrast to its extended pose in binding with hMAO-B (Figure 5A), 1l adopted a folded pose in the active site of hMAO-A (Figure 5B). The 1,4-benzodioxan moiety of this compound was located in the proximity of the FAD, and the N-phenyl ring pointed to the opposite side connected with the twisted carboxamide linker. The resulting folded pose might be due to the fact that hMAO-A has a single and smaller substrate cavity than hMAO-B.25 Compound 1l had to adopt a folded conformation in order to avoid structural conflicts with the bulky side chains of surrounding residues such as Tyr69, Phe208, and Phe352. Yet, the folded conformation of 1l would result in less-productive interactions with hMAO-A, which had been observed in other large hMAO-B-selective inhibitors.26 As compound 1a also exhibited lower binding free energy with hMAO-A, compared to that with hMAO-B (Table S1), we hypothesize that steric hindrance might play a critical role in preventing active compounds of this series from binding to hMAO-A.

Figure 5.

Figure 5

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(A) Docking pose of compound 1l in the active site of hMAO-B (PDB code: 2V5Z). (B) Docking pose of compound 1l in the active site of hMAO-A (PDB code: 2Z5X). Hydrogen bonds are shown as orange dotted lines. For clarity, only the relevant residue side chains are shown. FAD is rendered as white sticks, and compound 1l is rendered as yellow sticks.

To investigate the antineuroinflammatory effect of compound 1l, the inhibitory effects of 1l on the production of pro-inflammatory mediators were then evaluated. The levels of NO, TNF-α, and IL-1β were measured in LPS or Aβ1–42-stimulated BV2 cells with or without the tested compound. As illustrated in Figures 6A–C, LPS induced an obvious release of the pro-inflammatory mediators from BV2 cells. The levels of NO, TNF-α, and IL-1β were evaluated and determined to be enhanced by up to 11-fold, 6-fold, and 5-fold, respectively, compared to the control groups. While pretreating the BV2 cells with 1l at concentrations of 1 and 10 μM for 6 h, followed by the addition of LPS, the released levels of NO, TNF-α, and IL-1β from cells were reduced in a dose-dependent manner. Compared to the groups that were treated by LPS alone, ∼27%–41%, ∼15%–25%, and ∼31%–47% reductions in the release of NO, TNF-α, and IL-1β, respectively, were observed in LPS-stimulated BV2 cells when pretreated with 1l. We also investigated the inhibitory effects of compound 1l on the production of pro-inflammatory mediators in Aβ1–42-stimulated BV2 cells. As illustrated in Figures 6D–F, Aβ1–42 induced about 4-fold, 18.5-fold and 2.7-fold increase on the levels of NO, TNF-α and IL-1β respectively. While pretreating the BV2 cells with 1l at concentrations of 1, 5, and 10 μM for 6 h, followed by the addition of Aβ1–42, the released levels of NO, TNF-α, and IL-1β from cells were reduced in a dose-dependent manner. Compared to the Aβ1–42 alone groups treated, 15%–59%, 56%–76%, and 35%–58% reduction in the release of NO, TNF-α, and IL-1β, respectively, were observed in Aβ1–42-stimulated BV2 cells when pretreated with 1l. The above results clearly suggested that the hit compound 1l could effectively inhibit the release of NO, TNF-α, and IL-1β in both LPS- and Aβ1–42-stimulated BV2 cells, which indicated its potential to be developed as an antineuroinflammation agent.

Figure 6.

Figure 6

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Compound 1l inhibited the release of NO, TNF-α, and IL-1β in LPS- and Aβ1–42-stimulated BV2 cells. 0.5% DMSO acted as the vehicle control. (A–C) Depiction of the levels of NO, TNF-α, and IL-1β in LPS-stimulated BV2 cells with or without 1l. (D–F) Depiction of the levels of NO, TNF-α, and IL-1β in Aβ1–42-stimulated BV2 cells with or without 1l. The data are presented as mean ± SD of at least three independent experiments. [(*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. (a) versus control cells and (b) versus LPS or Aβ1–42 -treated cells.]

To investigate the neuroprotective effect of the potent compound 1l, the MTT assay was performed to evaluate the cell viability in the presence of Aβ1–42 with or without the tested compound. First, the cytotoxicity of compound 1l was determined in BV2 cells. As illustrated in Figure 7A, 1l exhibited little cytotoxicity in BV2 cells at the tested concentrations (1–10 μM). We then investigated the cytotoxicity induced by various concentrations of Aβ1–2 in BV2 cells. The results showed that Aβ1–42 reduced the cell viability in a dose-dependent manner, of which the percentage of viable cells was 57.1%, compared to the vehicle at 15 μM (Figure 7B). However, when pretreating with compound 1l at 0.01–1 μM for 24 h, the cell viability was recovered by 11%–39%, compared to the Aβ1–42-treated group (Figure 7C). These results suggest that 1l could attenuate the cytotoxicity induced by Aβ1–42 in the BV-2 cells. Taking account of the fact that 1l exhibit little cytotoxicity in SHSY5Y cells (0.01–50 μM, Figure 7D), we suppose the hit compound 1l could be developed as a potential neuroprotective agent with a wide safety window.

Figure 7.

Figure 7

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Cell viability of BV2 and SHSY5Y cells after treatment with or without compound 1l at different concentrations. 0.5% DMSO acted as the vehicle control. (A) Depiction of the cell viability of BV2 cells after treatment with 1l (1–10 μM) for 24 h. (B) Depiction of the cell viability of BV2 cells after treatment with various concentrations of Aβ1–42 (1–50 μM) for 24 h. (C) Depiction of the cell viability of BV2 cells treated with 15 μM Aβ1–42 with or without pretreatment with various concentrations of 1l (0.01–1 μM) for 24 h. (D) Depiction of the cell viability of SHSY5Y cells after treatment with 1l (0.01–50 μM) for 24 h. [(**) p < 0.01, (***) p < 0.001 versus vehicle cells or Aβ1–42-treated cells.]

In summary, a series of N-phenyl-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide derivatives were designed, synthesized, and evaluated for the inhibition of hMAO-B. SAR analysis suggested that the 1,4-benzodioxan moiety and the spatial position of the amide linker are important structural requirements for inhibitory potency. The introduction of halogen atoms, especially the chlorine atom, at the meta and para position on the N-phenyl ring would significantly increase the inhibitory activity. The most potent compound 1l found in this work exhibited an IC50 value of 0.0083 μM with an SI value of >4819, which was superior to that of rasagiline or safinamide. Kinetics and reversibility studies confirmed that compound 1l acted as a competitive and reversible inhibitor of hMAO-B. Moreover, 1l exhibited noticeable inhibitory effects on the releases of pro-inflammatory mediators NO, TNF-α, and IL-1β in the LPS or Aβ1–42-stimulated BV2 cells and attenuated the cytotoxicity induced by Aβ1–42 in BV2 cells. We believe our findings in this work offer an opportunity to search for novel potent MAO-B inhibitors with antineuroinflammatory activity, and the hit compound 1l constitutes a promising lead for further investigations.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant Nos. 81560562 and 21762055) and the Science and Technology Department of Guizhou Province (No. QKHPTRC-CXTD[2022]012).

Glossary

ABBREVIATIONS

MAO

monoamine oxidase

PD

Parkinson’s disease

AD

Alzheimer’s disease

LPS

lipopolysaccharide

Aβ

amyloid β

SAR

structure–activity relationship

SI

selectivity index

FAD

Flavin adenine dinucleotide

TNF-α

tumor necrosis factor α

IL-1β

interleukin-1β

MTT

(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00532.

  • Details of synthetic procedures, biochemical and biological assays, and analytical data (PDF)

Author Contributions

∇ Demeng Sun and Bo Wang contributed equally to this work. Demeng Sun, Bo Wang performed compound synthesis and biological assays. Yanmei Jiang, Zuo Kong contributed to compound synthesis. Mengxue Mu, Changhuan Yang contributed to biological assays. Jingbo Tan contributed to the X-ray diffraction analysis. Yun Hu supervised the study, and drafted and finalized the manuscript. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ml3c00532_si_001.pdf (6MB, pdf)

References

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