Discovery of a Potent and Selective MAO‐B Inhibitor From a Donepezil‐Linked Chalcone Library With Promising Antiparkinsonian Activity

ABSTRACT

A focused library of 19 donepezil‐linked chalcones (DLCs) was efficiently synthesised through microwave‐assisted Claisen‐Schmidt condensation and subsequently profiled for their inhibitory activities against cholinesterases (AChE and BuChE) as well as monoamine oxidases (MAO‐A and MAO‐B). The DLCs exhibited potent and selective inhibition of MAO‐B, with IC₅₀ values ranging from 0.019 to 18.98 μM, whereas activity toward MAO‐A was moderate to low (IC₅₀ = 0.81 to > 20 μM). Among the tested DLCs, DLC9 and DLC14 showed the highest MAO‐B inhibitory potential with IC₅₀ values of 0.054 ± 0.004 μM and 0.019 ± 0.0015 μM, respectively, and high selectivity indexes (> 370 and > 1052, respectively), whereas DLC12 displayed notable MAO‐A inhibition (IC₅₀ = 0.81 ± 0.035 μM). Kinetic and reversibility studies revealed that the selected two lead DLCs (DLC9 and DLC14) acted as mixed‐type reversible MAO‐B inhibitors, with Kᵢ values of 20.0 ± 2.83 nM and 10.0 ± 2.82 nM, respectively. Furthermore, IC₅₀ values of AChE inhibitory activities ranged from 5.40 to > 40 µM, whereas those of BuChE inhibitory activity range from 4.30 to > 40 µM. DLC6 showed the best AChE inhibitory potential with IC₅₀ values of 5.40 ± 0.29 µM, while DLC13 revealed effective BuChE inhibitory potential with an IC₅₀ value of 4.30 ± 0.89 µM. Molecular docking studies performed on hMAO‐A and hMAO‐B revealed that DLC14 establishes favourable π–π stacking within the aromatic cage of hMAO‐B and maintains complementary hydrophobic contacts along the substrate cavity, whereas DLC6 lacks this key interaction due to steric interference of the ethoxy substituent. Among the three most potent MAO‐B inhibitors (DLC2, DLC9, and DLC14), DLC2 exhibited the most favourable microsomal stability with the longest half‐life and lowest intrinsic clearance, whereas DLC14 showed comparable metabolic profiles in rat and human liver microsomes. Experimental BBB permeability assays were hindered by compound‐membrane interactions; however, in silico predictions indicated satisfactory oral bioavailability and brain penetration for all three candidates. In the MPTP‐induced rat model of parkinsonism, the selective MAO‐B inhibitor DLC14 and the non‐selective inhibitor DLC6 significantly improved motor deficits and behavioural impairments across open field, pole, bar, rotarod, and forced swim tests, with progressive improvements observed up to Day 28. Notably, DLC14 consistently outperformed DLC6 and demonstrated an efficacy profile comparable to that of Selegiline, highlighting its therapeutic potential as an antiparkinsonian agent. These results indicate that DLC14 is potent and selective MAO‐B inhibitor and could serve as promising candidate for the treatment of neurodegenerative disorders, such as Parkinson's disease.

1. Introduction

Neurodegenerative diseases (NDs) are some of the most important health problems of this century. (Lamptey et al. 2022) They are becoming worse since the population is getting older and there are no effective treatments that change the progression of the disease. Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis are just a few of the neurodegenerative disorders that have a huge impact on society and continue to drive drug discovery and development. (Choonara et al. 2009) Both social and economic impacts of NDs are substantial, and the World Health Organization (WHO) has designated NDs as a global health priority. (GBD Nervous System Disorders 2024) Among all NDs, PD is a devastating neurodegenerative disorder and the second most common neurodegenerative disease after AD, affecting more than 1% of the elderly population. The global number of PD cases has increased markedly since 1990 and is projected to reach millions more by 2050. (Aarsland et al. 2021; Su et al. 2025) According to GBD 2021 data and recent modelling studies, the prevalence of PD is expected to increase by more than 70‐100% between 2021 and 2050, with population aging identified as the major driving force behind this rise. (Zhu et al. 2024) Clinically, PD is a slowly progressive but highly multidimensional syndrome characterised not only by motor symptoms such as resting tremor, bradykinesia, rigidity, and postural instability, but also by premotor and non‐motor manifestations including hyposmia, constipation, sleep disturbances, autonomic dysfunction, depression, and cognitive impairment. In particular, cognitive decline and progression to dementia substantially contribute to the devastating impact of the disease on quality of life. (Jankovic and Tan 2020; Silva et al. 2022) The heterogeneity and complexity of PD are attributed to the interplay among genetic risk factors, environmental influences, and multiple molecular pathways. (Billingsley et al. 2018; Xu et al. 2023) Interconnected mechanisms such as α‐synuclein aggregation, mitochondrial dysfunction, impaired proteostasis, neuroinflammation, oxidative stress, and alterations in the gut‐brain axis severely limit the effectiveness of single‐target therapeutic approaches (Katsoulaki et al. 2025).

Current therapies mainly provide symptomatic relief through modulation of the dopaminergic system; however, no approved disease‐modifying treatment is currently available. The repeated failure of numerous clinical candidates, together with the slow pace and high attrition rates of conventional single‐target drug development, has increased interest in alternative strategies such as drug repurposing and multifunctional drug design. (Bashir et al. 2025; Cheong et al. 2019) Druggable genes and Mendelian randomization approaches facilitate the identification of genetically supported therapeutic targets, while network‐based systems genetics approaches have revealed PD‐associated pathways and potentially repurposable drugs such as simvastatin. (Storm et al. 2021) Considering the multifactorial nature of PD, multifunctional drug design aimed at the simultaneous modulation of multiple pathological targets within a single molecule has emerged as a feasible and increasingly adopted strategy for the development of future disease‐modifying therapies. (Katsoulaki et al. 2025) Accordingly, multifunctional drug design has gained prominence as a conceptual approach in medicinal chemistry that seeks to integrate many pharmacophoric aspects into a single molecule. Lead compounds obtained from this approach, termed multi‐target‐directed ligands (MTDLs), are engineered to concurrently affect many pathways. (Hossain and Hussain 2025; Turgutalp and Kizil 2024) Despite PD being the second most common neurodegenerative disorder, the number of truly disease‐modifying candidates advancing to late‐stage (Phase II‐III) clinical trials remains limited. Moreover, many current therapeutic efforts still predominantly focus on symptomatic motor control rather than neuroprotection or the management of non‐motor manifestations. (McFarthing et al. 2020; McFarthing et al. 2023) To overcome these limitations and accelerate PD drug discovery, there has been a growing transition from the classical “one drug‐one target” paradigm toward multitarget therapeutic strategies that more accurately reflect the multifactorial pathophysiology of PD. This complex pathology involves interconnected processes including dopaminergic neuronal loss, oxidative stress, neuroinflammation, mitochondrial and lysosomal dysfunction, metal dyshomeostasis, and pathological protein aggregation. (Boulaamane et al. 2023) Within this polypharmacology framework, molecular hybridization is increasingly used to design single molecules that combine structural and functional elements addressing several PD‐relevant pathways. (Sasaki and Sonnet 2021) Such hybrid molecules and MTDLs can be developed either de novo or through the recombination of pharmacophoric elements derived from approved dopaminergic, antidiabetic, or other CNS‐active drugs, as well as failed clinical candidates and bioactive natural products such as curcuminoids and dihydrochalcones. Their design is frequently supported by in silico methodologies including pharmacophore modelling, QSAR, molecular docking, network pharmacology, and graph neural network‐based approaches. (Chhabra et al. 2025; Katsoulaki et al. 2025) Collectively, these strategies aim to generate more effective, disease‐modifying therapies for PD by simultaneously modulating multiple interconnected mechanisms that drive neurodegeneration.

In cholinergic synapses, neurotransmission ceases by the enzymatic hydrolysis of acetylcholine (ACh) into acetate and choline, a process facilitated by cholinesterases (ChEs), specifically acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). (Reid et al. 2013) AChE is the major enzyme in a healthy adult brain. It breaks down around 80% of ACh, while BuChE breaks down the other 20%, mostly as a support role. (Giacobini 1992) At present, only six drugs have received approval from the U.S. Food and Drug Administration (FDA) for the symptomatic treatment of AD (Figure 1). Donanemab and lecanemab are monoclonal antibodies that specifically target amyloid‐β (Aβ) aggregates in the brain. (Sims et al. 2023) Memantine, on the other hand, works against N‐methyl‐d‐aspartate (NMDA) receptors. (Witt et al. 2004) Donepezil is a very selective AChE inhibitor, rivastigmine inhibits both AChE and BuChE, (Bar‐On et al. 2002) and galantamine inhibits AChE selectively while also modulating nicotinic acetylcholine receptors allosterically. (Greenblatt et al. 1999) Together, these drugs show the different ways that doctors are now using drugs to treat the symptoms and causes of AD. These agents constitute the current first‐line pharmacological interventions for patients with mild to severe stages of AD. Tacrine, the first AChE inhibitor to be licensed by the U.S. FDA in 1993, was subsequently withdrawn from the market in 2013 owing to hepatotoxicity and other adverse events. (Watkins 1994) Although the available drugs provide partial symptomatic relief, particularly with respect to cognitive impairment, they lack disease‐modifying efficacy and thus fail to prevent or slow the inexorable progression of AD (Sharma 2019).

Figure 1.

FDA approved drugs for the treatment of Alzheimer's disease.

Monoamine oxidases (MAOs) require flavin adenine dinucleotide (FAD) for their enzymatic activity. They adhere to the outside of the mitochondria and are present in significant amounts in the brain and gastrointestinal tract. They facilitate the oxidative deamination of several endogenous neurotransmitters and exogenous amines, playing a crucial role in regulating neurotransmitter metabolism and the metabolism of dietary amines. (Santin et al. 2021) In humans, two isoforms of the enzyme, MAO‐A and MAO‐B, have been identified. (Hong and Li 2019) These isoforms possess generally analogous sequences; nonetheless, they exhibit significant variability in substrate affinity, inhibitor sensitivity, and tissue distribution. MAO‐A metabolises serotonin and norepinephrine more efficiently, while MAO‐B preferentially interacts with phenylethylamine and benzylamine. Both isoforms promote the breakdown of dopamine. MAO activity is crucial for maintaining monoaminergic signalling within the central nervous system. Dysregulation of this enzyme pathway is significantly linked to the etiology of various neuropsychiatric and neurodegenerative disorders, particularly depression, AD, and PD. (Finberg and Rabey 2016; Kalimon et al. 2023; Ramsay and Albreht 2021) Importantly, the human brain has a high expression of mitochondrial‐bound MAO‐B, and there is mounting evidence that abnormal MAO‐B activity plays a role in neurodegenerative processes by catabolising monoamines excessively, which damages neurones. (Jones and Raghanti 2021; Tripathi and Ayyannan 2019; Yeung et al. 2019) Pharmacological inhibition of MAO‐B is therefore regarded as a promising treatment approach for reducing and delaying the course of neurodegeneration.

The complex and multifactorial pathogenesis of PD has positioned MTDLs, capable of simultaneously modulating multiple interconnected pathological pathways, as a rational and increasingly attractive strategy in medicinal chemistry. By moving beyond the limitations of current symptomatic therapies, this approach offers a promising framework for the development of novel therapeutic candidates with the potential not only to alleviate symptoms but also to slow or modify disease progression. (Koszła et al. 2021; Vijiaratnam et al. 2021) From a structural design perspective, MTDLs can be broadly classified into three major categories. In the linked approach, separate pharmacophoric moieties are covalently joined by a chemical linker; the length, flexibility, and physicochemical characteristics of the linker significantly influence biological activity, selectivity, and pharmacokinetic profile. The fused approach embodies an intermediate design, wherein separate pharmacophoric units are combined into a single continuous structure absence of an explicit linker, resulting in a hybridised framework that retains critical recognition motifs while reducing molecular size and conformational flexibility. In the merged (or overlap) approach, two or more pharmacophoric moieties are integrated into a single molecular scaffold, thereby yielding a compact structure in which the different recognition elements are embedded within the same framework. The three design methodologies, depicted schematically in Figure 2, form the conceptual basis for the systematic development of MTDLs. (Bajad et al. 2024; Pathak and Kabra 2024) A particularly compelling strategy combines ChE inhibition with monoamine oxidase (MAO) blockade. MAO‐B is the more critical of the two isoforms since it not only controls the breakdown of biogenic amines but also helps generate reactive oxygen species, which increases oxidative stress and damage to neurones. (Gaweska and Fitzpatrick 2011; Yeung et al. 2019) To counteract these pathogenic pathways, researchers have engineered new structures that can block both ChEs and MAOs. (Fonseca et al. 2017) These agents with dual inhibitory effects on ChE and MAO have been developed to simultaneously enhance cholinergic neurotransmission. (El‐Damasy et al. 2023; Mathew et al. 2021b) Carradori et al. reported the rational design and synthesis of multifunctional compounds exhibiting dual MAO‐B/AChE inhibitory activity together with antioxidant properties, highlighting their potential application in neurodegenerative disorders (Carradori et al. 2018). Natural‐product‐derived scaffolds have also attracted considerable attention in MAO‐targeted drug design, as demonstrated by De Monte et al. who investigated chemically modified constituents of Crocus sativus as human monoamine oxidase inhibitors through biological and molecular modelling studies (De Monte et al. 2014).

Figure 2.

The three design methodologies for designing MTDLs.

2. Results and Discussion

2.1. Design Strategy and Chemistry

Donepezil is known as a highly selective AChE inhibitor for the treatment of mild to moderate AD. The 5,6‐dimethoxy‐1‐indanone part of donepezil has been shown to interact with the peripheral anionic site of AChE. (Zhang et al. 2020) This interaction is very important for therapy since AChE not only helps break down acetylcholine but also helps toxic complexes form with Aβ peptides. (Alvarez et al. 1995) On the other hand, chalcones scaffold, belonging to the class of α,β‐unsaturated carbonyl compounds, has emerged as a versatile framework in modern drug discovery and is recognised as a privileged structural motif in medicinal chemistry. (Rajendran et al. 2022; Rammohan et al. 2020) Researchers have investigated into chalcone derivatives as MAO‐B inhibitors for PD. Chimenti et al. demonstrated that chalcone derivatives constitute a valuable scaffold for the development of monoamine oxidase inhibitors. (Chimenti et al. 2009) They have shown that they are very selective for MAO‐B over MAO‐A and that they can affect Aβ aggregation, tau protein aggregation, and neuroinflammation. This means that they can target many of the disease's bad effects. (Thapa et al. 2021) Recent studies in the literature on the chalcones incorporating heterocyclic motif such as morpholine, piperidine, imidazole, and piperazine have recognised them as potent and selective MAO‐B inhibitors, so establishing the chalcone containing heterocyclic motif as an optimal pharmacophore for MAO‐B inhibition. (Bajad et al. 2024; El‐Damasy et al. 2023; Lee et al. 2024; Mathew et al. 2021b; Osmaniye et al. 2018; Polo et al. 2019; Sasidharan et al. 2021b) In particular, morpholine‐containing chalcones have demonstrated remarkable potency and selectivity toward MAO‐B inhibition. Similarly, piperazine‐based derivatives have been identified as highly potent and selective MAO‐B inhibitors at the sub micromolar level. (El‐Damasy et al. 2023; Mathew et al. 2021a; Sasidharan et al. 2021a) In contrast, chalcones incorporating an imidazole ring generally exhibit inhibitory activity against both MAO‐A and MAO‐B isoforms, although their selectivity is comparatively lower than that observed for morpholine‐ or piperazine‐containing analogues, even among the most active derivatives. (Sasidharan et al. 2018) More recently, piperidine‐based chalcones have also attracted considerable attention as MAO‐B inhibitors. Several piperidinyl‐chalcone derivatives have exhibited MAO‐B inhibitory activity in the nanomolar range, highlighting their promising pharmacological potential. (Łażewska et al. 2026) Comparative evaluation of these scaffolds suggests that morpholine‐ and piperazine‐containing chalcones generally display superior potency, often with sub micromolar or even lower IC₅₀ values accompanied by high selectivity toward MAO‐B, (El‐Damasy et al. 2023; Mathew et al. 2021b; Sasidharan et al. 2021b) whereas imidazole derivatives tend to act as less selective but still pharmacologically relevant dual MAO inhibitors. (Sasidharan et al. 2018) Piperidine‐based chalcones, on the other hand, appear particularly attractive because of their dual‐target potential and, in some studies, have demonstrated comparable or even superior inhibitory potency. (Łażewska et al. 2026) Collectively, the available evidence indicates that chalcones containing morpholine or piperazine represent highly efficient MAO‐B inhibitory scaffolds, while imidazole derivatives are more appropriately regarded as dual MAO‐targeting agents. Meanwhile, piperidine‐based chalcones have emerged as promising next‐generation multitarget ligands with potential applications in neurodegenerative disease therapeutics.

Through the linking of a donepezil scaffold to a chalcone with heterocyclic motif, we suggest building target molecules that combine the complementary pharmacological characteristics of donepezil and chalcone, thereby enabling simultaneous inhibition of ChEs and MAOs. This multi‐target approach will have synergistic effects, increasing the therapeutic potential to treat complex and multifactorial diseases such as AD. Figure 3 shows the rationale behind the design of the donepezil‐linked chalcones (DLCs). The donepezil‐derived moiety is expected to preserve its strong affinity for the catalytic active site of AChE. To effectively modulate monoamine metabolism, it is necessary to inhibit selective MAO‐B, and the heterocyclic structure has been proven to be an essential pharmacophoric component for this purpose. (El‐Damasy et al. 2023; Jeong et al. 2021; Lee et al. 2024; Mathew et al. 2021b; Sasidharan et al. 2021b) The goal of creating these DLCs is to treat more effectively by combining two or more of these features into one chemical structure that acts as an inhibitor of both CHE and MAO. The chalcone framework has two roles: it acts as a structural linker that connects the donepezil core to the piperidine ring, and it also acts as a pharmacophoric component that could make the drug more effective. The conjugated α,β‐unsaturated carbonyl framework of the chalcone makes the structure more rigid and planar. This may have helped the two pharmacophores fit together better for better binding. The more expansive π‐system also makes the electronic communication inside the molecule better, which could make it easier for enzymes to interact with the active regions.(Agoni et al. 2022; Malik et al. 2022).

Figure 3.

The three design methodologies for designing MTDLs.

The design of the DLCs was guided by strategic alterations to the indanone fragment from a structure‐activity relationship (SAR) perspective. The fundamental structure of donepezil comprises an indanone molecule featuring two methoxy substituents. This work methodically modified these substituents by including several atom groups with differing quantities, types, and positional arrangements to assess their impact on biological potency. The modifications aimed to investigate the influence of electronic and steric variables on the structural integrity of the indanone framework, ensuring it remains sufficiently robust to inhibit enzyme activity. Moreover, the piperidine moiety at the para‐position in the ring was selected toward MAO‐B selectivity throughout the series. This is due to previous studies demonstrating that this heterocyclic structure is a crucial pharmacophoric feature for selective MAO‐B inhibition. (Deshmukh et al. 2025; Marcel Gomes et al. 2017) This design method facilitated the exact optimisation of AChE contacts through diverse substitution(s) at various positions, while maintaining the MAO‐B inhibitory function of the heterocycle‐chalcone unit, ultimately aiming for dual ChE/MAO activity. A targeted library of DLCs was created by systematically varying the indanone fragment while preserving the piperidine‐based chalcone moiety at the para‐position as a consistent pharmacophore for MAO‐B inhibition. The indanone ring was diversified by using mono, di, and trisubstituents in various positional configurations to investigate the impact of electronic and steric changes. This library thus offers a systematic approach to investigate SARs for dual ChE/MAO inhibition and to discover optimised scaffolds with potential therapeutic significance for AD.

The Claisen‐Schmidt condensation is still one of the most common ways to make chalcones because it is very good at making new carbon‐carbon bonds. This transformation is a distinct variant of the crossed aldol condensation, wherein the α‐carbon of an enolisable ketone reacts with the carbonyl carbon of an aromatic aldehyde to generate an α,β‐unsaturated ketone framework, such as a chalcone. The production of the desired DLCs (DLC1‐19) followed to the pathway defined in Scheme 1. The reaction is commonly conducted under acidic or basic catalysis in polar solvents with moderate heating (50°C–100°C), providing a direct method for synthesising chalcone scaffolds. (Gomes et al. 2017) In an effort to improve reaction efficiency and reduce environmental impact, microwave‐assisted methodologies have been developed as greener alternatives. Under these conditions, electron‐rich acetophenone derivatives (containing mono‐, di‐, or trisubstituents) reacted with 4‐(1‐piperidinyl)benzaldehyde in the presence of ethanolic solution of sodium hydroxide (40%) at 100°C to afford DLCs in high efficiency and shorter reaction times (Choudhary et al. 2025).

Scheme 1.

Synthesis of donepezil‐linked chalcone analogues (DLC1‐19).

The structures of all freshly synthesised DLCs were characterised by FT‐IR and NMR spectroscopy, in conjunction with High Resolution Mass Spectrometry (HRMS) for molecular mass determination. In the FTIR spectra, all compounds exhibited characteristic absorption bands corresponding to aromatic C─H stretching vibrations in the region of approximately 3019–3100 cm⁻¹, whereas the aliphatic C─H stretching frequencies of the piperidine and alkoxy substituents appeared between 2828 and 2999 cm⁻¹. The formation of the α,β‐unsaturated ketone framework was verified by the presence of strong carbonyl stretching bands observed around 1626–1659 cm⁻¹, consistent with conjugated chalcone systems. The ¹H NMR spectra of all synthesised compounds further supported the proposed structures. The α‐ and β‐olefinic protons of the chalcone moiety appeared as two characteristic doublets with large coupling constants, confirming the trans (E)‐configuration of the enone system. Generally, the β‐proton resonated at a more downfield region compared with the α‐proton due to the deshielding effect of the conjugated carbonyl group. Aromatic proton signals were observed in their expected regions depending on the electronic nature and substitution pattern of the phenyl rings. Methoxy substituents gave characteristic singlet signals at approximately δ 3.7–4.0 ppm, while ethoxy and butoxy derivatives displayed the expected quartet‐triplet and multiplet patterns corresponding to OCH₂ and terminal alkyl protons. The methylenedioxy analogue (DLC12) exhibited a distinct singlet at δ 6.05 ppm attributable to the OCH₂O bridge. Signals corresponding to the piperidine ring protons were consistently observed in the aliphatic region, typically around δ 3.1–3.4 ppm for the N‐adjacent methylene groups and δ 1.4–1.8 ppm for the remaining methylene protons. The ¹³C NMR spectra were also in good agreement with the proposed structures. The conjugated carbonyl carbon atoms of the chalcone scaffold were detected in the range of δ 188–201 ppm. Signals attributed to aromatic methoxy carbons were generally observed around δ 55–56 ppm, whereas alkoxy methylene carbons appeared at slightly lower fields depending on the substituent type. The aromatic and olefinic carbon resonances were distributed in the expected regions according to the electronic properties of the substituents attached to the phenyl rings. Furthermore, the piperidine methylene carbons were consistently observed around δ 24–49 ppm. Finally, HRMS analyses confirmed the molecular compositions of all synthesised compounds.

2.2. Enzyme Inhibition Assay

2.2.1. Inhibitory Activities Against Monoamine Oxidase Enzymes (MAO‐A/B)

MAO inhibitory activities of the 19 compounds in the DLC series were measured following the standard protocol described in the materials and methods section. Preliminary screening against both MAO‐A and MAO‐B were carried out at a 10 µM concentration of compounds. Results revealed that two DLCs (DLC3 and DLC12) showed less than 50% residual activities towards MAO‐A, whereas six of them (DLC1‐3, DLC9, DLC14, and DLC15) revealed no residual activity towards MAO‐B at the given concentration (Table 1). Further, concentration‐dependent inhibitory activities of lead compounds as well as other potential molecules were evaluated and IC₅₀ values were calculated. The IC₅₀ values for MAO‐A inhibition ranged from 0.81 to > 20 µM, while those for MAO‐B inhibition ranged from 0.019 to 18.98 µM. Among the obtained DLCs, DLC9 and DLC14 revealed the most potent towards MAO‐B with IC₅₀ values of 0.054 ± 0.004 µM and 0.019 ± 0.0015, respectively, whereas DLC12 showed the highest potency towards MAO‐A with an IC₅₀ value of 0.81 ± 0.035 μM (Table 1 and Figure 4). Additionally, DLC1‐3, DLC5, DLC8, DLC12, and DLC19 exhibited IC₅₀ values in the range of 0.096 to 0.692 μM towards MAO‐B. (Table 1). Furthermore, all the tested DLCs were highly selective towards MAO‐B, particularly DLC9 and DLC14 with selectivity index (SI) values of > 370 and > 1052, respectively. When compared with the reference compounds, DLC14 was comparable to or slightly more potent than safinamide (0.021 ± 0.002 µM) towards MAO‐B.

Table 1.

Residual activities and IC₅₀ values of DLCs with MAO‐A and MAO‐B. ^a

ID	% Residual activity (10 µM)		IC50 (µM)		SIb
	MAO‐A	MAO‐B	MAO‐A	MAO‐B
DLC1	67.82	ND	> 20	0.425 ± 0.062	> 47.58
DLC2	65.21	ND	> 20	0.096 ± 0.005	> 208.33
DLC3	48.69	ND	9.72 ± 0.39	0.197 ± 0.001	49.34
DLC4	50.43	63.85	15.62 ± 1.71	2.97 ± 0.418	5.26
DLC5	85.21	13.25	> 20	0.692 ± 0.08	> 29.90
DLC6	49.56	79.51	11.21 ± 0.34	18.98 ± 0.994	0.59
DLC7	81.41	84.34	> 20	11.74 ± 1.176	> 1.70
DLC8	61.06	3.61	> 20	0.662 ± 0.095	> 30.21
DLC9	85.84	ND	> 20	0.054 ± 0.004	> 370.37
DLC10	86.72	13.25	> 20	4.422 ± 0.37	> 4.73
DLC11	91.92	16.87	> 20	2.39 ± 0.03	> 8.36
DLC12	19.46	20.48	0.81 ± 0.035	0.32 ± 0.075	2.53
DLC13	65.85	8.42	> 20	0.597 ± 0.0144	> 33.50
DLC14	83.89	ND	> 20	0.019 ± 0.0015	> 1052.63
DLC15	79.67	ND	> 20	0.931 ± 0.0426	> 21.48
DLC16	86.17	28.42	> 20	9.97 ± 1.037	> 2.00
DLC17	86.99	45.26	> 20	11.42 ± 3.38	> 1.75
DLC18	86.99	6.31	> 20	2.92 ± 0.55	> 6.84
DLC19	81.3	1.05	> 20	0.507 ± 0.046	> 39.44
Safinamide				0.021 ± 0.0006
Pargyline				0.138 ± 0.004
Toloxatone			1.67 ± 0.15
Clorgyline			0.0085 ± 0.0008

Abbreviation: ND, not detected.

^a Results were expressed as mean standard deviation (SD) of triplicate experiments.

^b SI values were expressed for MAO‐B, compared with MAO‐A, based on their IC₅₀ values.

Figure 4.

Concentration‐dependent inhibition curves of MAO‐B by (A) DLC14 and (B) DLC9. The results were expressed as mean ± SD of triplicate experiments.

2.2.2. Inhibitory Activities Against Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE)

AChE and BuChE inhibitory activities of the DLCs were analysed following the standard protocol described in the materials and methods section. Preliminary screening against AChE and BuChE were carried out at a 40 μM concentration of the DLCs. Results revealed that three DLCs (DLC3, DLC5, and DLC6) showed potential inhibitory activity towards AChE, whereas three of them (DLC3, DLC5, DLC11, and DLC13) were highly potent towards BuChE (Table 2). Further, concentration‐dependent inhibitory activities were evaluated, and IC₅₀ values were calculated. The IC₅₀ values for AChE inhibition ranged from 5.40 to 7.53 μM, while those for BuChE inhibition ranged from 4.30 to 6.46 μM. Among the tested DLCs, DLC6 was the most potent inhibitor of AChE with an IC₅₀ value of 5.40 ± 0.29 µM, followed by DLC3 and DLC5 with IC₅₀ values of 7.53 ± 0.29 and 6.49 ± 0.10 µM, respectively. In contrast, DLC13 was the most potent inhibitor of BuChE with an IC₅₀ value of 4.30 ± 0.89 µM, followed by DLC3 and DLC5 with IC₅₀ values of 6.46 ± 1.00 and 6.07 ± 0.49 µM, respectively (Table 2).

Table 2.

Residual activities and IC₅₀ values of DLCs against AChE and BuChE. ^a

ID	% Residual activity (40 µM)		IC50 (µM)
	AChE	BuChE	AChE	BuChE
DLC1	71.90 ± 13.26	51.43 ± 6.27	> 40	38.63 ± 1.25
DLC2	58.91 ± 14.16	25.58 ± 3.41	> 40	18.01 ± 0.98
DLC3	ND	1.92 ± 1.31	7.53 ± 0.29	6.46 ± 1.00
DLC4	51.61 ± 12.9	18.48 ± 6.38	> 40	14.80 ± 1.86
DLC5	ND	ND	6.49 ± 0.10	6.07 ± 0.49
DLC6	ND	42.15 ± 2.60	5.40 ± 0.29	33.06 ± 1.91
DLC7	56.19 ± 7.56	42.52 ± 6.92	> 40	28.10 ± 4.01
DLC8	49.78 ± 8.81	37.68 ± 2.10	> 40	25.91 ± 1.71
DLC9	55.07 ± 3.22	18.26 ± 1.03	> 40	15.01 ± 0.62
DLC10	31.58 ± 9.91	ND	23.60 ± 1.82	14.44 ± 0.11
DLC11	70.77 ± 10.32	16.24 ± 4.72	> 40	8.46 ± 1.03
DLC12	42.19 ± 15.82	55.61 ± 0.08	21.26 ± 0.73	> 40
DLC13	14.52 ± 8.14	ND	16.08 ± 1.65	4.30 ± 0.89
DLC14	82.74 ± 0.65	36.93 ± 3.65	> 40	26.74 ± 0.83
DLC15	21.76 ± 3.65	48.17 ± 7.06	10.98 ± 1.07	> 40
DLC16	31.53 ± 7.21	26.90 ± 1.07	17.31 ± 0.52	28.96 ± 0.56
DLC17	37.86 ± 9.16	57.14 ± 11.06	> 40	> 40
DLC18	16.42 ± 2.53	28.61 ± 4.67	11.17 ± 0.89	24.68 ± 0.40
DLC19	35.99 ± 2.18	77.44 ± 3.31	> 40	> 40
Donepezil			0.0064 ± 0.0001	0.148 ± 0.006

Abbreviation: ND, not detected.

^a Results were expressed as mean standard deviation (SD) of triplicate experiments.

2.2.3. Structure‐Activity Relationships (SAR) Analysis

Nineteen structurally different DLCs were evaluated for their MAO‐A, MAO‐B, and cholinesterase inhibitory activities. The A ring of chalcone refers to the phenyl ring linkage with the carbonyl group, whereas the B ring refers to the benzene ring connected to the double bond. The SAR was investigated by modification of various positions (mono‐ or polysubstituted) with different group(s) in the A ring of the chalcone scaffold, since the same substituent at different positions may alter the activity. The best inhibitory potential of DLC14 towards MAO‐B was attributed to the 3,5‐disubstitution of methoxy groups in the A ring, followed by the 2,5‐disubstitution in DLC9. Our results revealed that the 3,5‐dimethoxy substitution in DLC14 increased MAO‐B inhibitory activity by fivefold when compared to the mono‐substituted methoxy group at the para‐position in DLC2 (Figure 5). Similarly, the 2,5‐disubstituted methoxy groups in DLC9 increased the activity by 7.8‐fold compared to the mono‐substituted methoxy group at the meta‐position in DLC1. Interestingly, when compared with 2,4‐dimethoxy substituted DLC analogue, the 2,5‐ and 3,5‐substitutions enhanced the inhibitory potential by ~12‐ and ~35‐fold, respectively, clearly indicating that methoxy groups at the meta‐position to each other were more effective than para‐substitutions for MAO‐B inhibition. Furthermore, in AChE and BuChE inhibitory activities, DLC6 with an ethoxy substituent at the para‐position of the A ring showed the highest AChE inhibition, whereas DLC3 with a methoxy group at the same position was comparatively less potent. Our results demonstrated that chain elongation from methoxy to ethoxy at the para‐position increased AChE inhibition by 1.2‐fold, suggesting that subtle structural modifications of the chalcone scaffold strongly influence the potency and selectivity of enzyme inhibition.

Figure 5.

SAR analysis of MAO‐B inhibitors based on the DLCs.

2.2.4. Enzyme Kinetic Studies

Kinetics analysis of the two lead compounds DLC9 and DLC14 towards MAO‐B was carried out at three different concentrations of each inhibitor (~ 0.5, 1.0, and 2.0 times of their IC₅₀ values) against five different concentrations of benzylamine as a substrate. Lineweaver‐Burk (LB) plots of the inhibitors revealed that both DLC9 and DLC14 act as mixed type inhibitors. Furthermore, secondary plot showed the K _i values of 20.0 ± 2.83 and 10.0 ± 2.82 nM, respectively, for DLC9 and DLC14 (Figure 6).

Figure 6.

Lineweaver‐Burk (LB) plots for MAO‐B inhibition of DLC14 (A) and DLC9 (C) and respective secondary plots (i.e., slopes vs inhibitor concentrations) of DLC14 (B) and DLC9 (D). The experiments were carried out at five different concentrations of a substrate benzylamine and three different concentrations of DLC9 or DLC14. These results were expressed as mean ± SD of triplicate experiments.

2.2.5. Reversibility Studies

The reversibility of MAO‐B inhibition by DLC9 and DLC14 was evaluated by dialysis methods described in the materials and methods section. The concentrations of each inhibitor (DLC9 and DLC14) used were approximately double of their IC₅₀ values. The enzyme activity recoveries were determined by comparing relative undialyzed (A_U) and dialyzed (A_D) residual activities. The relative activity of DLC14 was recovered from 26.12% (A_U) to 63.19% (A_D), whereas DLC9 was recovered from 29.23% (A_U) to 73.03% (A_D). The recoveries of both DLCs were comparable to that of the reference compound safinamide (17.00%–80.33%) but were clearly distinguished from that of pargyline (17.01%–18.90%) (Figure 7). These results revealed that both DLC9 and DLC14 are reversible inhibitors of MAO‐B.

Figure 7.

Recovery of MAO‐B inhibition by DLC9 and DLC14 using dialysis experiments. The concentration of DLC9 and DLC14 used were approximately double of their IC₅₀ values. After a 30 min of preincubation, the mixtures were dialyzed for 6 h with a buffer change at 3‐h interval. The results were expressed as mean ± SD of triplicate experiments.

2.2.6. Molecular Docking Studies

We selected both DLC6 and DLC14 to study the interaction with hMAO‐A and hMAO‐B at atomistic level through molecular docking simulation using autodock‐4.2 with ADT in MGLTools‐1.5.7. X‐ray crystallographic structure of hMAO‐A (PDB:2BXR) and hMAO‐B (PDB:2BYB) were used for the purpose and the simulation was done by adopting the protocol reported by our group earlier (Jayaprakash et al. 2008).

2.2.6.1. Interaction of DLC14 With hMAO‐B (Figure 8A)

Figure 8.

3D‐interaction plot of A. DLC14 with hMAO‐B, B. DLC6 with hMAO‐B, C. DLC14 with hMAO‐A, and D. DLC6 with hMAO‐A. All the interacting residues (gold), cofactor (green) and compounds (sky blue) represented as tubes with atoms coloured by heteroatom. Hydrophobic interactions depicted as red lines, hydrogen bonding interaction in blue dashed line and π‐π interaction as gray tube connecting centroid of the aromatic rings (A displays π–π stacking interaction between DLC14 and TYR398, B displays T‐shaped interaction between DLC6 and TYR326).

Phenyl ring carrying 3,5‐dimethoxy substitution was accommodated well inside the aromatic cage (formed by FAD, TYR398, and TYR435) due to the π‐π stacking interaction with TYR398. Rest of the molecule was well accommodated in the narrow linear tunnel and showing hydrophobic interaction with the residues lining the tunnel. Vinylic region has shown hydrophobic interaction with LEU171, while the phenyl ring attached with it established the hydrophobic interaction with PHE168, LEU171, and ILE199. The piperidyl extension has shown hydrophobic interaction with ILE199 and ILE316.

2.2.6.2. Interaction of DLC6 with hMAO‐B (Figure 8B)

The orientation of DLC6 is quite similar to DLC14 with predominantly hydrophobic interactions with same residues. The major difference being that the phenyl ring carrying 4‐ethoxy functional group was pushed away from the aromatic cage as the bulkier and flexible ethoxy group occupied that region. This has led to the loss of π‐π stacking interaction that deemed to be crucial for potency of the compound.

2.2.6.3. Interaction of DLC14 with hMAO‐A (Figure 8C)

The hMAO‐A has a larger pocket volume with three hydrophobic pockets. It has an aromatic cage similar to hMAO‐B formed by FAD, TYR407, and TYR444 (Pocket 1). The following residues carves the pocket 2: GLY71, GLY74, ARG206, ILE207, PHE208, GLU216, and TRP441, while the following residues carves pocket 3: ILE180, ILLE335, LEU337, MET350, and PHE352. Larger pocket volume makes it difficult for smaller molecules to establish tight interaction with the active site residues. DLC14 orients in such a way that pocket 1 is kept unoccupied. The phenyl ring carrying 3,5‐dimethoxy substitution positioned in way to show hydrophobic interaction with pocket 3 residue, PHE352 and pocket 1 residue, TYR407. A hydrogen bonding interaction also observed between methoxy oxygen of DLC14 and hydroxyl hydrogen of TYR407. The second phenyl ring carrying piperidyl substitution engaged with pocket 2 residue, TRP441 through hydrophobic interaction. This has kept the unsaturated carbonyl linker over pocket 1 like a flap covering it and engaged with TYR444 through a hydrophobic interaction.

2.2.6.4. Interaction of DLC6 with hMAO‐A (Figure 8D)

Orientation and interactions were strikingly similar to DLC14. Pocket 3 accommodated the ethoxy functional group and engaged with ILE335, LEU337, and PHE352 through hydrophobic interaction. The interaction analysis clearly shows that unoccupied aromatic cage and absence of π‐π stacking interaction with TYR residues of aromatic cage in both hMAO‐A and hMAO‐B led to the reduction in affinity and hence potency.

DLC6 was a potent and selective inhibitor of AChE, with an IC₅₀ value of 5.40 µM and a selectivity index of approximately 6 (BuChE/AChE). At the concentrations required to inhibit hMAO‐A and hMAO‐B (IC₅₀ values of 11.21 and 18.98 µM, respectively), DLC6 would also inhibit AChE, with narrower selectivity indices of approximately 2 (hMAO‐A/AChE) and 3.5 (hMAO‐B/AChE). Thus, DLC6 appears to be a truly multi‐targeted ligand, and we selected it for further investigation of its interactions with hAChE (PDB: 7D9O) and hBuChE (PDB: 7AWH).

2.2.6.5. Interaction of DLC6 With hAChE (Figure 9A)

Figure 9.

3D‐interaction plot of A. DLC6 with hAchE, B. DLC6 with hBuChE, All the interacting residues (khaki), and compounds (gray) are represented as tubes with atoms coloured by heteroatom. Hydrophobic interactions depicted as orang dashed lines, hydrogen bonding interaction in blue dashed lines and π‐π interaction as yellow tube connecting centroid (silver coloured sphere) of the aromatic rings (A displays π–π stacking interaction between DLC6 and TYR337, B displays T‐shaped interaction between DLC6 and HIS438). Label colors: Catalytic triad in blue, choline binding site in pink, acyl binding site in orange and peripheral anionic site in red.

Human acetylcholinesterase has a deep, narrow gorge with five distinct sites that interact with specific regions of the substrate acetylcholine: the catalytic triad (SER203, GLU334, HIS447), acyl binding site (TRP236, PHE295, PHE297), oxyanion hole (GLY121, GLY122, ALA204), choline binding site (TRP86, TYR133, TYR337, PHE338), and peripheral anionic site (TYR72, ASP74, TYR124, TRP286, TYR341). DLC6 orients within the narrow gorge such that the phenyl ring bearing the piperidine moiety forms a π–π stacking interaction with TYR337, along with hydrophobic interactions with residues of the choline binding site. This orientation places the piperidine ring closer to HIS447, a residue of the catalytic triad, enabling hydrophobic interaction. A hydrogen‐bonding interaction was observed between the carbonyl oxygen of DLC6 and the backbone NH of PHE295, an acyl binding site residue. The phenyl ring carrying the ethoxy substituent establishes hydrophobic interactions with residues of the peripheral anionic site. No interaction with residues of the oxyanion hole was observed.

2.2.6.6. Interaction of DLC6 With hBuChE (Figure 9B)

Human butyrylcholinesterase has a similar deep gorge, with a catalytic triad (SER198, GLU325, HIS438), acyl binding site (TRP231, LEU286, VAL288), oxyanion hole (GLY116, GLY117, ALA199), choline binding site (TRP82, TYR128, ALA328, PHE329), and peripheral anionic site (ASN68, ASP70, GLN119, ALA277, TYR341). Substitution of PHE295 → LEU286, PHE297 → VAL288, TYR337 → ALA328, TYR72 → ASN68, and TRP286 → ALA277 in BuChE makes its acyl binding site, choline binding site, and peripheral anionic site more spacious than those of AChE. DLC6 interacts with BuChE in a reverse orientation, placing the piperidine ring closer to the peripheral anionic site but without forming any interaction there. The carbonyl oxygen of DLC6 forms hydrogen‐bonding interactions with the sidechain OH of SER198 and the imidazole NH of HIS438, both of which are catalytic triad residues. In addition, the phenyl ring carrying the ethoxy substituent establishes a T‐shaped interaction with HIS438. No interaction was observed with residues forming either the oxyanion hole or the peripheral anionic site. The phenyl ring bearing the piperidine moiety shows hydrophobic interaction with LEU286, an acyl binding site residue, and is unfavourably positioned near the polar residue THR120. These features result in weaker interaction with BuChE than with AChE, which may explain the higher IC₅₀ values.

2.2.7. In Vitro Metabolic Stability and Permeability Studies

Top three (DLC2, DLC9, and DLC14) potent and selective inhibitors of MAO‐B were selected for in vitro metabolic stability study using rat and human liver microsomes. Only DLC14 has shown a metabolic stability profile that is similar in both rat and human liver microsomes with a half‐life (t_1/2) of 4.5 min and clearance (CL_int) of 301–310 µL/min/mg‐protein (Table 3). DLC9 displayed a poor half‐life with higher clearance in both rat and human liver microsomal fractions, while DLC2 had the best half‐life of 8.15 min with low clearance of 172 µL/min/mg‐protein.

Table 3.

In vitro metabolic stability studies using rat and human liver microsomal fractions.

Compound	Rat liver microsomes			Human liver microsomes
	% metabolism in 30 min	t1/2 (min)	CLint (µL/min/mg protein)	% metabolism in 30 min	t1/2 (min)	CLint (µL/min/mg protein)
DLC2	100	1.65	844	87	8.15	172
DLC9	100	1.85	756	100	1.05	1346
DLC14	100	4.6	310	98	4.6	302
VER	90	8.8	158	91	8.6	161

Abbreviation: VER, Verapamil used as positive control.

Blood‐Brain Barrier (BBB) permeability assessment was done for DLC2, DLC9, and DLC14 using PAMPA with Carbamazepine as positive control. While Carbamazepine's data matches with the historical data (Pe (x10⁻⁶) cm/sec, 11.9, high), the other three test compounds failed due to extensive binding with lipid membrane on the donor side in the absence (Table 4) as well as presence of bovine serum albumin (0.1% and 1%). Hence, the experiment was repeated with MDCK‐MDR1 cell monolayer with Digoxin as positive control. We encountered similar problem with MDCK‐MDR1 cell monolayer too and hence the values calculated may not be reliable. As per the data presented in Table 5, DLC14 was having low permeability.

Table 4.

Blood brain barrier permeability study using PAMPA.

Compound ID	Pe (x10−6) cm/sec	% Retention*
DLC2	NR	100
DLC9	NR	84
DLC14	NR	90
Carbamazepine (50 µM)	11.9	12

Note: Pe(x10⁻⁶) cm/sec < 1 ‐ Low; 1‐10 ‐ Medium; > 10 – High.

^*binding to the BBB membrane, NR‐Not reportable.

Table 5.

Blood brain barrier permeability study using MDCK‐MDR1.

Compound ID	Mean Papp A to B	Mean Papp B to A	Efflux Ratio	Rank	Note
DLC2	0	0	0	NA	Test item adhering to the monolayer
DLC9	3	2.1	0.7	Moderate	Test item adhering to the monolayer
DLC14	0.3	0.1	0.4	Low	Test item adhering to the monolayer
Digoxin	0.3	2.0	5.7	High

Abbreviation: NA, not applicable.

As in vitro results generated were not reliable due to compounds adherence to the monolayer, we predicted permeability characteristics of the compounds through Deep‐PK server (https://biosig.lab.uq.edu.au/deeppk/). All the three compounds were predicted to have good oral bioavailability and BBB permeability.

2.2.8. Pharmacology

The potent and selective MAO‐B inhibitor (DLC14) and the non‐selective inhibitor MAO inhibitor (DLC6) were selected for assessing their potential as antiparkinsonian agents. Parkinsonism was induced chemically in rats using the neurotoxin MPTP, (Smeyne and Jackson‐Lewis 2005) subsequently both the negative control and treatment groups were subjected to open field test, (Gould et al. 2009) bar test, (Fischer et al. 2002) pole test, (Krout et al. 2024) forced swim test, (Lino‐de‐Oliveira et al. 2005) and rotarod test (Shiotsuki et al. 2010) on day 7, 14, and 28 (Figures 10, 11, 12).

Figure 10.

Open field test in rats. Control‐Positive control received only normal saline, MPTP (30 mg/kg)‐Negative control received MPTP 30 mg/kg body weight i.p. once daily for 5 days, SEL (10 mg/kg), DLC6 (10 mg/kg), DLC14 (10 mg/kg)‐Treatment groups received MPTP 30 mg/kg body weight + respective drugs 10 mg/kg body weight for five days and respective drugs 10 mg/kg body weight for additional two days. SEL: Selegiline. Sample size: 5 animals in a group (n = 5), Statistics: One‐way ANOVA followed by TukeyHSD: * (p), ** (p $\le$ 0.01), *** (p $\le$ 0.001), **** (p $\le$ 0.0001), ^ns (p $\ge 0.05$).

Figure 11.

Pole test in rats. Control‐Positive control received only normal saline, MPTP (30 mg/kg)‐Negative control received MPTP 30 mg/kg body weight i.p. once daily for 5 days, SEL (10 mg/kg), DLC6 (10 mg/kg), DLC14 (10 mg/kg)‐Treatment groups received MPTP 30 mg/kg body weight + respective drugs 10 mg/kg body weight for 5 days and respective drugs 10 mg/kg body weight for additional 2 days. SEL: Selegiline. Sample size: five animals in a group (n = 5), Statistics: One‐way ANOVA followed by TukeyHSD: * (p $\le 0.05$), ** (p $\le$ 0.01), *** (p $\le$ 0.001), **** (p $\le$ 0.0001), ^ns (p $\ge 0.05$).

Figure 12.

Catalepsy time in seconds (Bar test in rats), Fall latency in seconds (Rotarod test in rats) and Swim time in seconds (Forced swim test in rats). Control‐Positive control received only normal saline, MPTP (30 mg/kg)‐Negative control received MPTP 30 mg/kg body weight i.p. once daily for 5 days, SEL (10 mg/kg), DLC6 (10 mg/kg), DLC14 (10 mg/kg)‐Treatment groups received MPTP 30 mg/kg body weight + respective drugs 10 mg/kg body weight for 5 days and respective drugs 10 mg/kg body weight for additional 2 days. SEL: Selegiline. Sample size: five animals in a group (n = 5), Statistics: One‐way ANOVA followed by TukeyHSD: * (p $\le 0.05$), ** (p $\le$ 0.01), *** (p $\le$ 0.001), **** (p $\le$ 0.0001), ns (p $\ge 0.05$).

Open field test (OFT): Six parameters: number of line crossing (count), immobility time (s), rearing (count), number of centre zone entries (count), time spent in centre (s) and grooming episodes (count) were recorded for all the groups. Increase in number of line crossings and decrease in immobility time inferred as an improvement in locomotor activity. Increase in number of centre zone entries and rearing inferred as an improvement in curiosity, motivation and exploratory behaviour. Increase in number of centre zone entries and time spent in centre inferred as anxiolytic behaviour. Finally, reduction in grooming episodes inferred as reduction of stress and stereotype behaviour. All these parameters were recorded on Day 7, Day 14, and Day 28 for assessing progressive improvement. In case of line crossing both DLC6 and DLC14 exhibited a progressive improvement up to Day 14 and a significant decrease on Day 28, but reduction in immobility time has shown progressive decrease till Day 28 (Supporting Information Figure S58A and S58B, Table S1). This clearly shows a progressive improvement in locomotor activity. Comparison of Day 14 data revealed that Selegiline, DLC6, and DLC14 treatment groups were showing significant improvement (p < 0.0001) in locomotor activity in comparison with negative control. Number of centre zone entries and rearing shows a steady increase till Day 28, but the difference was significant only up to Day 14 in case of DCL6 (p < 0.05) and not significant after Day 7 in case of DLC14 (p = ns) (Supporting Information Figure S58A and S58B, Table S1). Comparison of Day 14 data suggested a clear case of significant improvement (p < 0.0001) in exploratory behaviour for treatment groups (Selegiline, DLC6, and DLC14) in comparison with negative control. For both DLC6 and DLC14, the data for time spent in centre zone was progressive till Day 14 and became stabilized after Day 14 (Supporting Information Figure S58A and S58B, Table S1). On Day 14, all the three treatment groups (Selegiline, DLC6, and DLC14) have shown a significant improvement (p < 0.0001) in anxiolytic behaviour. The inference also considered increase in number of centre zone entries for the treatment groups. In case of grooming episodes, there was no significant difference between Day 7 through Day 28 for DLC6, but there was a significant difference (p < 0.05) between Day 7 and Day 28 for DLC14 (Supporting Information Figure S58A and S58B, Table S1). On Day 14, the treatment groups displayed a significant improvement (Selegiline: p < 0.0001, DLC6 and DLC14: p < 0.01) in comparison with Negative control. Overall, locomotor activity, exploratory, and anxiolytic behaviour improved in the treatment groups and the order of effectivity found to be Selegiline > DLC14 > DLC6. In case of reduction in stress and stereotyping all the treatment groups performed equally.

The Pole Test was used to measure the bradykinesia (slowness of movement) and motor coordination in rats. Two parameters: T‐turn and T‐total were recorded for all the groups. A progressive decrease (p < 0.0001) in both the counts was observed for DLC6 as well as DLC14 (Supporting Information Figure S62, Table S5). All the three treatment groups have shown a significant (p < 0.0001) decrease in both the counts in comparison with negative control on Day 14, a clear indication of improvement in movement disorder. The order of effectivity was found to be similar to the one observed with OFT i.e. Selegiline > DLC14 > DLC6.

Bar test was performed to assess the decrease in catalepsy time(s) in treatment group in comparison with negative control. Catalepsy is a neurological state characterised by muscle rigidness and fixed posture with decreased responsiveness and movement. It's a key symptom of catatonia associated with neurological conditions like parkinsonism. DLC6 shows a significant increase (p < 0.001) between Day 7 and 14, followed by significant decrease (p < 0.0001) between Day 14 and 28, while a progressive decrease (p < 0.0001) was observed for DLC14 till Day 14 and gets stabilized after that (Supporting Information Figure S59, Table S2). All the three treatment groups displayed significant decrease (p < 0.0001) in comparison with negative control on Day 14 and the order of effectiveness was found to be Selegiline > DLC14 > DLC6.

Rotarod test was done to determine the fall latency(s), time in seconds that an animal stays on a rotating rod before falling. It is used as a measure of motor coordination, balance and learning ability. Both the compounds, DLC6 and DLC14, have shown a progressive increase (p < 0.0001) in fall latency (s) through Day 7 to Day 28 (Supporting Information Figure S61, Table S4). The treatment groups displayed significant increase in duration (p < 0.0001) in comparison with negative control on Day 14 and the order of effectiveness was found to be similar to the one observed for bar test (Selegiline > DLC14 > DLC6).

Forced swim test was performed to estimate the swim time(s) as a measure of akinesia (lack of movement due to motor deficit) and depressive like state often associated with parkinsonism. A progressive increase (p < 0.0001) in swim time(s) was observed for both DLC6 and DLC14 through Day 7 to Day 28 (Supporting Information Figure S60, Table S3). A significate increase (p < 0.0001) in swim time(s) was observed for all the three treatment groups and was complementing the observation with immobility(s) in OFT. The order of effectiveness was found to be Selegiline > DLC14 > DLC6.

A significant improvement was observed for treatment groups in comparison with the negative control clearly showing the recovery in behavioural and motor symptoms associated with parkinsonism. The order of effectiveness was found to be Selegiline > DLC14 > DLC6. A ~ 1000‐fold difference in potency was observed for DLC6 in comparison with DLC14 in case of MAO‐B inhibition (Table 1), yet it was found to be equally effective as DLC14. This may be attributed to its non‐selective inhibition of MAO isoforms, where MAO‐A inhibition may cause alleviation of depressive‐like symptoms leading to its effectiveness equivalent to DLC14.

3. Conclusions

In conclusion, we have designed and synthesised a focused library of 19 chalcone molecules (DLCs) using microwave‐assisted Claisen‐Schmidt condensation. We have also comprehensively assessed their inhibitory profiles against MAO‐A, MAO‐B, AChE, and BuChE. The analysis of the structure‐activity relationship demonstrated that the potency and selectivity of MAO‐B were significantly improved by specific methoxy substitutions on the A ring, particularly at the 2,5‐ and 3,5‐positions, while MAO‐A displayed minimal activity. DLC9 and DLC14 were the most potent MAO‐B inhibitors in the series, with nanomolar IC₅₀ values of 54 ± 4 nM and 19 ± 1.5 nM, respectively, and extraordinarily high SI (> 370 and > 1052, respectively). The kinetic characterisation and reversibility investigations have confirmed that both compounds function as mixed‐type reversible inhibitors, with Kᵢ values of 10.0 ± 2.82 nM and 20.0 ± 2.8 nM, respectively. This confirms a strong and specific interaction with the enzyme active site. Conversely, the cholinesterase inhibition was moderate, with DLC6 and DLC13 exhibiting the highest AChE and BuChE activity, respectively. Taken together, these findings establish DLC9 and DLC14 as potent and selective MAO‐B inhibitors, offering exciting potential building blocks for therapeutic medications aimed at neurodegenerative diseases like Parkinson's. They also show how important strategic methoxy substitution is for controlling the enzyme's selectivity and activity. Overall, the docking analysis highlights the critical role of aromatic cage occupancy and π‐π stacking interactions with TYR residues in governing MAO inhibitory potency, with DLC14 demonstrating a more optimal binding profile than DLC6, particularly toward hMAO‐B. Collectively, DLC2 emerged as the most metabolically stable derivative, while computational ADME profiling supported the potential CNS exposure of the series despite experimental limitations in permeability assessment. These findings warrant further optimization to balance metabolic stability and reliable brain penetration characteristics. Collectively, the in vivo findings establish DLC14 as a promising lead candidate with robust antiparkinsonian activity, producing significant improvements in locomotor function, motor coordination, bradykinesia, and affective behaviour. Its superior efficacy over the non‐selective analogue DLC6 further underscores the pharmacological advantage of selective MAO‐B inhibition in the management of Parkinsonian symptoms.

4. Experimental

4.1. Chemicals and Apparatus

All reagents and solvents used in this study were of analytical grade (≥ 95% purity) and purchased from commercial suppliers, unless otherwise specified, without further purification. Recombinant MAO‐A and MAO‐B, kynuramine, benzylamine, safinamide, pargyline, toloxatone, and clorgyline were purchased from sigma (St. Louis, MO, USA). Anhydrous mono‐ and dibasic sodium phosphate were purchased from Daejung Chemicals and Metals (Siheung, South Korea). A DiaEasy dialyzer (6‐8 kDa) was purchased from BioVision (St. Grove MA, USA). All other chemicals used in this experiment were of analytical grade. Reactions were carried out using an Anton Paar Monowave 400 Microwave Synthesis Reactor (Graz, Austria) in sealed G30 vials with magnetic stirring, under the specified conditions of temperature and time. Column chromatography was performed on silica gel 60 (63‐200 μm particle size) using suitable solvent systems as eluents, while reaction progress was monitored by thin‐layer chromatography (TLC) on silica gel 60 F₂₅₄ aluminium plates (Merck) and visualized under UV light (254 or 365 nm). Melting points (mp) were determined with a Büchi B‐540 apparatus and are reported uncorrected. Infrared (IR) spectra were recorded on an FTIR spectrometer equipped with a diamond ATR module. ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectra were obtained on a Bruker Avance III HD spectrometer using CDCl₃ as the solvent and tetramethyl silane (TMS) as the internal standard. Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz. High‐resolution mass spectra (HRMS) were acquired on a Waters SYNAPT G1 mass spectrometer (Waters, USA).

4.2. Animals

Adult Wistar rats weighing between 200 and 220 g were used for the experiment. The animals were obtained from the Laboratory Animal House, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India, on 25.06.2025, under Protocol No. 1972/PH/BIT/121/25/IAEC. The animals were maintained under standard laboratory conditions, with controlled temperatures (24°C–27°C), humidity (60% ± 15%), and a 12‐h light/dark cycle. They were allowed free access to standard food and water and were acclimated for 7 days prior to the experiment. All experiments were conducted in accordance with the guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India.

4.3. General Procedure for the Synthesis of Target Chalcones

A mixture of 4‐(1‐piperidinyl)benzaldehyde (2.46 mmol) and the corresponding substituted acetophenone (2.46 mmol) was dissolved in absolute ethanol (5 mL) in a sealed G30 reaction vial (Anton Paar, Graz, Austria) equipped with a silicone septum, snap cap, and magnetic stirring at 600 rpm. To this solution, 40% NaOH (0.675 mL) was added at room temperature. The reaction vial was then placed in an Anton Paar Monowave 400 Microwave Synthesis Reactor (Graz, Austria) and heated to 90°C within 1 min, followed by irradiation at 100°C for 6 min. Upon completion, the temperature was reduced to 55°C in the reactor and the reaction mixture was subsequently cooled to room temperature (or placed in a refrigerator if necessary). The resulting precipitate was collected by filtration, washed with cold hexane, and dried. The crude product was further purified by silica gel column chromatography using an appropriate solvent system as the eluent to afford the desired DLC analogues, when required.

4.3.1. (E)‐1‐(2‐Methoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC1)

Using the general procedure, DLC1 was prepared from 2’‐methoxyacetophenone and isolated as a reddish brown oil. Yield: 51%. FTIR (ATR) ν (cm⁻¹): 3073, 3003 (CH_aromatic), 2934, 2851 (CH_aliphatic), 1649 (> C = O), 1593, 1578, 1512, 1483, 1462, 1452, 1433, 1385, 1335, 1277, 1236, 1180, 1161, 1125, 1111, 1059, 1016. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.53–7.48 (m, 2H, β‐H and CH_aromatic), 7.34 (d, J = 8.9 Hz, 2H, CH_aromatic), 7.33‐7.29 (m, 1H, CH_aromatic), 7.13 (d, J = 15.7 Hz, 1H, α‐H), 6.91 (t, J = 7.5 Hz, 1H, CH_aromatic), 6.85 (d, J = 8.4 Hz, 1H, CH_aromatic), 6.72 (d, J = 8.9 Hz, 2H, CH_aromatic), 3.71 (s, 3H, OCH₃), 3.11 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.58‐1.42 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 192.83 (> C = O), 157.79, 152.95, 144.18, 132.29, 130.09, 129.95, 129.86, 124.20, 122.88, 120.53, 114.65, 111.66 (C_aromatic and C_olefinic), 55.62 (OCH₃), 48.82 (CH₂N_piperidine), 25.36 (CH₂CH₂N_piperidine), 24.27 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₁H₂₄NO₂ [M + H]⁺: 322.1807; found: 322.1807.

4.3.2. (E)‐1‐(3‐Methoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC2). (Elkhalifa et al. 2020)

Using the general procedure, DLC2 was prepared from 3’‐methoxyacetophenone and isolated as a brown solid. Yield: 41%, mp 97.1–97.6°C. FTIR (ATR) ν (cm⁻¹): 3084 (CH_aromatic), 2922, 2847 (CH_aliphatic), 1651 (> C = O), 1570, 1518, 1483, 1462, 1447, 1429, 1387, 1319, 1304, 1261, 1244, 1221, 1186, 1167, 1125, 1030. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.77 (d, J = 15.5 Hz, 1H, β‐H), 7.58 (d, J = 7.6 Hz, 1H, CH_aromatic), 7.56–7.51 (m, 3H, CH_aromatic), 7.39 (t, J = 7.9 Hz, 1H, CH_aromatic), 7.33 (d, J = 15.5 Hz, 1H, α‐H), 7.13–7.09 (m, 1H, CH_aromatic), 6.89 (d, J = 8.8 Hz, 2H, CH_aromatic), 3.88 (s, 3H, OCH₃), 3.32 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.73–1.60 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 190.41 (> C = O), 159.83, 153.22, 145.49, 140.39, 130.29, 129.42, 124.35, 120.88, 118.76, 117.85, 114.75, 112.79 (C_aromatic and C_olefinic), 55.47 (OCH₃), 49.03 (CH₂N_piperidine), 25.45 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₁H₂₄NO₂ [M + H]⁺: 322.1807; found: 322.1807.

4.3.3. (E)‐1‐(4‐Methoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC3).(Nelson et al. 2013)

Using the general procedure, DLC3 was prepared from 4’‐methoxyacetophenone and isolated as a brown solid. Yield: 35%, mp 135.9‐136.3°C. FTIR (ATR) ν (cm⁻¹): 3074 (CH_aromatic), 2930, 2837 (CH_aliphatic), 1647 (> C = O), 1595, 1578, 1560, 1508, 1449, 1427, 1387, 1341, 1308, 1260, 1188, 1163, 1125, 1022. ¹H NMR (600 MHz, CDCl ₃ ): δ 8.03 (d, J = 8.9 Hz, 2H, CH_aromatic), 7.77 (d, J = 15.5 Hz, 1H, β‐H), 7.54 (d, J = 8.7 Hz, 2H, CH_aromatic), 7.37 (d, J = 15.5 Hz, 1H, α‐H), 6.97 (d, J = 8.8 Hz, 2H, CH_aromatic), 6.89 (d, J = 8.7 Hz, 2H, CH_aromatic), 3.88 (s, 3H, OCH₃), 3.31 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.81–1.53 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 188.91 (> C = O), 163.05, 153.11, 144.54, 131.78, 130.59, 130.10, 124.64, 117.64, 114.85, 113.70 (C_aromatic and C_olefinic), 55.46 (OCH₃), 49.12 (CH₂N_piperidine), 25.47 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₁H₂₄NO₂ [M + H]⁺: 322.1807; found: 322.1808.

4.3.4. (E)‐1‐(4‐(Methylthio)phenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC4).(Elkhalifa et al. 2020)

Using the general procedure, DLC4 was prepared from 4’‐(methylthio)acetophenone and isolated as a brown solid. Yield: 18%, mp 146.0‐146.5°C. FTIR (ATR) ν (cm⁻¹): 3032 (CH_aromatic), 2932, 2851 (CH_aliphatic), 1645, 1620 (> C═O), 1584, 1570, 1547, 1514, 1447, 1429, 1387, 1354, 1285, 1240, 1194, 1179, 1125, 1090, 1022, 1005. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.95 (d, J = 8.5 Hz, 2H, CH_aromatic), 7.77 (d, J = 15.5 Hz, 1H, β‐H), 7.54 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.35 (d, J = 15.5 Hz, 1H, α‐H), 7.30 (d, J = 8.5 Hz, 2H, CH_aromatic), 6.89 (d, J = 8.8 Hz, 2H, CH_aromatic), 3.31 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 2.53 (s, 3H, SCH₃), 1.72‐1.60 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 189.34 (> C═O), 153.17, 145.10, 144.77, 135.23, 130.23, 128.82, 125.13, 124.43, 117.45, 114.77 (C_aromatic and C_olefinic), 49.05 (CH₂N_piperidine), 25.45 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine), 14.92 (SCH₃). HRMS(+ ESI) m/z calcd for C₂₁H₂₄NOS [M + H]⁺: 338.1579; found: 338.1579.

4.3.5. (E)‐1‐(2‐Ethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC5)

Using the general procedure, DLC5 was prepared from 2’‐ethoxyacetophenone and isolated as an orange solid. Yield: 19%, mp 94.4–95.2°C. FTIR (ATR) ν (cm⁻¹): 3075, (CH_aromatic), 2980, 2932, 2853 (CH_aliphatic), 1643 (> C═O), 1597, 1578, 1512, 1487, 1474, 1381, 1329, 1279, 1221, 1198, 1184, 1165, 1117, 1040, 1020. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.60 (dd, J = 7.6, 1.8 Hz, 1H, CH_aromatic), 7.55 (d, J = 15.8 Hz, 1H, β‐H), 7.46 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.40 (td, J = 8.5, 1.8 Hz, 1H, CH_aromatic), 7.26 (d, J = 15.7 Hz, 1H, α‐H), 7.03‐6.98 (m, 1H, CH_aromatic), 6.95 (d, J = 8.3 Hz, 1H, CH_aromatic), 6.86 (d, J = 8.9 Hz, 2H, CH_aromatic), 4.10 (q, J = 7.0 Hz, 2H, OCH₂), 3.28 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.72‐1.56 (m, 6H, CH₂CH₂N_piperidine), 1.40 (t, J = 7.0 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, CDCl ₃ ): δ 193.09 (> C═O), 157.33, 152.94, 143.71, 132.23, 130.27, 130.05, 129.96, 124.72, 123.32, 120.58, 114.83, 112.63 (C_aromatic and C_olefinic), 64.25 (OCH₂), 49.09 (CH₂N_piperidine), 25.45 (CH₂CH₂N_piperidine), 24.31 (CH₂CH₂CH₂N_piperidine), 14.85 (OCH₂CH₃). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₂ [M + H]⁺: 336.1964; found: 336.1966.

4.3.6. (E)‐1‐(4‐Ethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC6)

Using the general procedure, DLC6 was prepared from 4’‐ethoxyacetophenone and isolated as an orange solid. Yield: 49%, mp 145.2‐145.9°C. FTIR (ATR) ν (cm⁻¹): 3068 (CH_aromatic), 2976, 2934, 2837 (CH_aliphatic), 1647 (> C═O), 1597, 1508, 1450, 1427, 1389, 1339, 1304, 1258, 1188, 1161, 1117, 1020. ¹H NMR (600 MHz, CDCl ₃ ): δ 8.00 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.75 (d, J = 15.5 Hz, 1H, β‐H), 7.52 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.36 (d, J = 15.5 Hz, 1H, α‐H), 6.94 (d, J = 8.8 Hz, 2H, CH_aromatic), 6.88 (d, J = 8.9 Hz, 2H, CH_aromatic), 4.10 (q, J = 7.0 Hz, 2H, OCH₂), 3.29 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.72‐1.58 (m, 6H, CH₂CH₂N_piperidine), 1.44 (t, J = 7.0 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, CDCl ₃ ): δ 188.84 (> C═O), 162.44, 153.04, 144.40, 131.53, 130.54, 130.04, 124.63, 117.62, 114.82, 114.10 (C_aromatic and C_olefinic), 63.67 (OCH₂), 49.09 (CH₂N_piperidine), 25.44 (CH₂CH₂N_piperidine), 24.31 (CH₂CH₂CH₂N_piperidine), 14.69 (OCH₂CH₃). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₂ [M + H]⁺: 336.1964; found: 336.1967.

4.3.7. (E)‐1‐(4‐Butoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC7)

Using the general procedure, DLC7 was prepared from 4’‐butoxyacetophenone and isolated as an orange solid. Yield: 40%, mp 133.1–133.6°C. FTIR (ATR) ν (cm⁻¹): 3048 (CH_aromatic), 2965, 2940, 2855 (CH_aliphatic), 1639 (> C═O), 1599, 1576, 1508, 1474, 1450, 1435, 1418, 1393, 1339, 1308, 1250, 1223, 1211, 1171, 1024, 1003. ¹H NMR (600 MHz, CDCl ₃ ): δ 8.01 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.76 (d, J = 15.5 Hz, 1H, β‐H), 7.54 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.38 (d, J = 15.5 Hz, 1H, α‐H), 6.96 (d, J = 8.8 Hz, 2H, CH_aromatic), 6.89 (d, J = 8.8 Hz, 2H, CH_aromatic), 4.04 (t, J = 6.5 Hz, 2H, OCH₂), 3.31 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.83–1.77 (m, 2H, OCH₂CH₂), 1.73–1.60 (m, 6H, CH₂CH₂N_piperidine), 1.55–1.48 (m, 2H, CH₂CH₃), 0.99 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (150 MHz, CDCl ₃ ): δ 188.91 (> C═O), 162.71, 153.09, 144.42, 131.52, 130.58, 130.07, 124.70, 117.71, 114.87, 114.17 (C_aromatic and C_olefinic), 67.92 (OCH₂), 49.14 (CH₂N_piperidine), 25.47 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine), 31.19, 19.21 (CH₂CH₂), 13.82 (CH₃). HRMS(+ ESI) m/z calcd for C₂₄H₃₀NO₂ [M + H]⁺: 364.2277; found: 364.2277.

4.3.8. (E)‐1‐(2,4‐Dimethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC8)

Using the general procedure, DLC8 was prepared from 2’,4’‐dimethoxyacetophenone and isolated as a brown solid. Yield: 65%, mp 86.0‐86.4°C. FTIR (ATR) ν (cm⁻¹): 3086 (CH_aromatic), 2918, 2843 (CH_aliphatic), 1638 (> C═O), 1593, 1578, 1547, 1514, 1466, 1454, 1431, 1327, 1279, 1244, 1198, 1184, 1128, 1105, 1016. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.70 (d, J = 8.6 Hz, 1H, CH_aromatic), 7.62 (d, J = 15.7 Hz, 1H, β‐H), 7.48 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.31 (d, J = 15.7 Hz, 1H, α‐H), 6.87 (d, J = 8.8 Hz, 2H, CH_aromatic), 6.55 (dd, J = 8.6, 2.2 Hz, 1H, CH_aromatic), 6.49 (d, J = 2.1 Hz, 1H, CH_aromatic), 3.88 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 3.28 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.74‐1.59 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 191.00 (> C = O), 163.65, 160.07, 152.93, 143.10, 132.52, 129.97, 125.03, 123.28, 122.89, 114.93, 104.97, 98.74 (C_aromatic and C_olefinic), 55.76, 55.51 (2xOCH₃), 49.21 (CH₂N_piperidine), 25.48 (CH₂CH₂N_piperidine), 24.35 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₃ [M + H]⁺: 352.1913; found: 352.1928.

4.3.9. (E)‐1‐(2,5‐Dimethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC9)

Using the general procedure, DLC9 was prepared from 2’,5’‐dimethoxyacetophenone and isolated as a brown oil. Yield: 29%. FTIR (ATR) ν (cm⁻¹): 3065 (CH_aromatic), 2997, 2930, 2843 (CH_aliphatic), 1641, 1604 (> C═O), 1557, 1516, 1489, 1460, 1433, 1406, 1333, 1275, 1219, 1180, 1165,1117, 1034, 1020. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.56 (d, J = 15.8 Hz, 1H, β‐H), 7.46 (d, J = 8.7 Hz, 2H, CH_aromatic), 7.19 (d, J = 15.7 Hz, 1H, α‐H), 7.14 (d, J = 3.1 Hz, 1H, CH_aromatic), 6.98 (dd, J = 9.0, 3.1 Hz, 1H, CH_aromatic), 6.91 (d, J = 9.0 Hz, 1H, CH_aromatic), 6.85 (d, J = 8.8 Hz, 2H, CH_aromatic), 3.82 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.28 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.74‐1.54 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 192.84 (> C = O), 153.58, 153.09, 152.21, 144.58, 130.50, 130.20, 124.48, 122.89, 118.21, 114.77, 114.43, 113.42 (C_aromatic and C_olefinic), 56.58, 55.84 (2xOCH₃), 49.05 (CH₂N_piperidine), 25.45 (CH₂CH₂N_piperidine), 24.35 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₃ [M + H]⁺: 352.1913; found: 352.1914.

4.3.10. (E)‐1‐(2,6‐Dimethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC10)

Using the general procedure, DLC10 was prepared from 2’,6’‐dimethoxyacetophenone and isolated as a yellowish brown solid. Yield: 80%, mp 205.5–206.4°C. FTIR (ATR) ν (cm⁻¹): 3069 (CH_aromatic), 2999, 2940, 2924, 2847, 2833 (CH_aliphatic), 1626 (> C = O), 1580, 1560, 1520, 1468, 1458,1300, 1250, 1234, 1179, 1105, 1059, 1018. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.39 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.30 (t, J = 8.4 Hz, 1H, CH_aromatic), 7.22 (d, J = 16.0 Hz, 1H, β‐H), 6.84 (d, J = 8.8 Hz, 2H, CH_aromatic), 6.80 (d, J = 16.0 Hz, 1H, α‐H), 6.60 (d, J = 8.4 Hz, 2H, CH_aromatic), 3.76 (s, 6H, 2xOCH₃), 3.28 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.73‐1.56 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 195.30 (> C═O), 157.50, 153.10, 146.11, 130.35, 130.17, 124.87, 124.25, 119.02, 114.75, 104.11 (C_aromatic and C_olefinic), 55.96 (OCH₃), 49.09 (CH₂N_piperidine), 25.41 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₃ [M + H]⁺: 352.1913; found: 352.1913.

4.3.11. (E)‐1‐(3,4‐Dimethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC11). (Emam et al. 2021)

Using the general procedure, DLC11 was prepared from 3’,4’‐dimethoxyacetophenone and isolated as a brown oil. Yield: 66%. FTIR (ATR) ν (cm⁻¹): 3078 (CH_aromatic), 2932, 2837 (CH_aliphatic), 1647 (> C═O), 1580, 1508, 1462, 1450, 1427, 1416, 1385, 1300, 1258, 1236, 1182, 1125, 1020. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.78 (d, J = 15.4 Hz, 1H, β‐H), 7.67 (dd, J = 8.3, 1.8 Hz, 1H, CH_aromatic), 7.62 (s, 1H, CH_aromatic), 7.54 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.39 (d, J = 15.4 Hz, 1H, α‐H), 6.91 (d, J = 8.3 Hz, 1H, CH_aromatic), 6.88 (d, J = 8.8 Hz, 2H, CH_aromatic), 3.96 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.29 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.70‐1.59 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 188.71 (> C = O), 153.09, 152.84, 149.12, 144.55, 131.98, 130.13, 124.55, 122.65, 117.36, 114.80, 110.84, 109.99 (C_aromatic and C_olefinic), 56.05, 56.02 (2xOCH₃), 49.06 (CH₂N_piperidine), 25.45 (CH₂CH₂N_piperidine), 24.33 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₃ [M + H]⁺: 352.1913; found: 352.1912.

4.3.12. (E)‐1‐(Benzo[d][1,3]dioxol‐5‐yl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC12)

Using the general procedure, DLC12 was prepared from 3’,4’‐(methylenedioxy)acetophenone and isolated as an orange solid. Yield: 47%, mp 151.4–151.9°C. FTIR (ATR) ν (cm⁻¹): 3090 (CH_aromatic), 2918, 2837 (CH_aliphatic), 1641 (> C═O), 1599, 1560, 1514, 1499, 1437, 1389, 1356, 1310, 1242, 1182, 1125, 1109, 1018. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.76 (d, J = 15.4 Hz, 1H, β‐H), 7.63 (dd, J = 8.1, 1.1 Hz, 1H, CH_aromatic), 7.55–7.49 (m, 3H, CH_aromatic), 7.31 (d, J = 15.4 Hz, 1H, α‐H), 6.88 (d, J = 9.1 Hz, 3H, CH_aromatic), 6.05 (s, 2H, OCH₂O), 3.30 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.80‐1.53 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 188.41 (> C═O), 153.14, 151.25, 148.14, 144.84, 133.68, 130.16, 124.49, 124.26, 117.40, 114.80, 108.44, 107.83 (C_aromatic and C_olefinic), 101.74 (OCH₂O), 49.07 (CH₂N_piperidine), 25.46 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₁H₂₂NO₃ [M + H]⁺: 336.1600; found: 336.1600.

4.3.13. (E)‐1‐(2,3‐Dihydrobenzo[b][1,4]dioxin‐6‐yl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC13)

Using the general procedure, DLC13 was prepared from 1,4‐benzodioxan‐6‐yl methyl ketone and isolated as an orange solid. Yield: 78%, mp 138.6–139.1°C. FTIR (ATR) ν (cm⁻¹): 3078 (CH_aromatic), 2916, 2851 (CH_aliphatic), 1643, 1601 (> C═O), 1570, 1540, 1429, 1387, 1325, 1285, 1209, 1184, 1159, 1125. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.75 (d, J = 15.5 Hz, 1H, β‐H), 7.62–7.56 (m, 2H, CH_aromatic), 7.52 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.33 (d, J = 15.4 Hz, 1H, α‐H), 6.94 (d, J = 8.3 Hz, 1H, CH_aromatic), 6.88 (d, J = 8.8 Hz, 2H, CH_aromatic), 4.34–4.30 (m, 2H, OCH₂), 4.30–4.27 (m, 2H, OCH₂), 3.30 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.76–1.59 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 188.68 (> C = O), 153.12, 147.51, 144.68, 143.34, 132.65, 130.15, 124.56, 122.44, 117.89, 117.46, 117.16, 114.82 (C_aromatic and C_olefinic), 64.70, 64.18 (2xOCH₂), 49.09 (CH₂N_piperidine), 25.47 (CH₂CH₂N_piperidine), 24.35 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₂H₂₄NO₃ [M + H]⁺: 350.1756; found: 350.1754.

4.3.14. (E)‐1‐(3,5‐Dimethoxyphenyl)‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC14)

Using the general procedure, DLC14 was prepared from 3’,5’‐dimethoxyacetophenone and isolated as a brown solid. Yield: 58%, mp 84.3–84.9°C. FTIR (ATR) ν (cm⁻¹): 3100 (CH_aromatic), 2936, 2835 (CH_aliphatic), 1659 (> C═O), 1576, 1516, 1449, 1422, 1387, 1348, 1296, 1285, 1240, 1202, 1184, 1150, 1125, 1063, 1034. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.77 (d, J = 15.5 Hz, 1H, β‐H), 7.51 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.29 (d, J = 15.5 Hz, 1H, α‐H), 7.14 (d, J = 2.3 Hz, 2H, CH_aromatic), 6.86 (d, J = 8.9 Hz, 2H, CH_aromatic), 6.64 (t, J = 2.3 Hz, 1H, CH_aromatic), 3.84 (s, 6H, 2xOCH₃), 3.29 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.69‐1.58 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 190.16 (> C═O), 160.83, 153.21, 145.57, 141.02, 130.33, 124.25, 117.70, 114.70, 106.22, 104.55 (C_aromatic and C_olefinic), 55.60 (OCH₃), 48.97 (CH₂N_piperidine), 25.44 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₂H₂₆NO₃ [M + H]⁺: 352.1913; found: 352.1909.

4.3.15. (E)‐3‐(4‐(Piperidin‐1‐yl)phenyl)‐1‐(2,3,4‐trimethoxyphenyl)prop‐2‐en‐1‐one (DLC15)

Using the general procedure, DLC15 was prepared from 2’,3’,4’‐trimethoxyacetophenone and isolated as an orange solid. Yield: 75%, mp 123.6–124.3°C. FTIR (ATR) ν (cm⁻¹): 3075 (CH_aromatic), 2995, 2930, 2849 (CH_aliphatic), 1645 (> C═O), 1587, 1512, 1460, 1406, 1315, 1287, 1254, 1234, 1204, 1182, 1125, 1096, 1013. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.62 (d, J = 15.7 Hz, 1H, β‐H), 7.50 (d, J = 8.9 Hz, 2H, CH_aromatic), 7.44 (d, J = 8.7 Hz, 1H, CH_aromatic), 7.29 (d, J = 15.7 Hz, 1H, α‐H), 6.88 (d, J = 8.9 Hz, 2H, CH_aromatic), 6.74 (d, J = 8.8 Hz, 1H, CH_aromatic), 3.92 (s, 6H, 2xOCH₃), 3.90 (s, 3H, OCH₃), 3.30 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.75–1.55 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 191.26 (> C═O), 156.46, 153.43, 153.05, 144.14, 142.16, 130.13, 127.49, 125.50, 124.63, 122.54, 114.84, 107.22 (C_aromatic and C_olefinic), 62.10, 61.07, 56.10 (3xOCH₃), 49.11 (CH₂N_piperidine), 25.46 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₃H₂₈NO₄ [M + H]⁺: 382.2018; found: 382.2010.

4.3.16. (E)‐3‐(4‐(Piperidin‐1‐yl)phenyl)‐1‐(2,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (DLC16)

Using the general procedure, DLC16 was prepared from 2’,4’,5’‐trimethoxyacetophenone and isolated as a brown solid. Yield: 77%, mp 130.6–130.8°C. FTIR (ATR) ν (cm⁻¹): 3061 (CH_aromatic), 3001, 2930, 2843 (CH_aliphatic), 1636 (> C═O), 1599, 1553, 1541, 1508, 1470, 1398, 1346, 1327, 1269, 1252, 1213, 1194, 1180, 1138, 1057, 1022. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.67 (d, J = 15.6 Hz, 1H, β‐H), 7.50 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.44 (d, J = 15.6 Hz, 1H, α‐H), 7.36 (s, 1H, CH_aromatic), 6.87 (d, J = 8.9 Hz, 2H, CH_aromatic), 6.54 (s, 1H, CH_aromatic), 3.95 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.28 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.70–1.59 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 190.16 (> C = O), 154.37, 153.02, 152.91, 143.31, 142.90, 129.98, 125.04, 123.11, 121.19, 114.90, 113.22, 97.39 (C_aromatic and C_olefinic), 56.93, 56.32, 56.11 (3xOCH₃), 49.16 (CH₂N_piperidine), 25.46 (CH₂CH₂N_piperidine), 24.33 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₃H₂₈NO₄ [M + H]⁺: 382.2018; found: 382.2018.

4.3.17. (E)‐3‐(4‐(Piperidin‐1‐yl)phenyl)‐1‐(2,4,6‐trimethoxyphenyl)prop‐2‐en‐1‐one (DLC17)

Using the general procedure, DLC17 was prepared from 2’,4’,6’‐trimethoxyacetophenone and isolated as a brown oil. Yield: 76%. FTIR (ATR) ν (cm⁻¹): 3078 (CH_aromatic), 2926, 2841 (CH_aliphatic), 1636 (> C═O), 1582, 1512, 1452, 1412, 1385, 1223, 1204, 1180, 1153, 1121, 1082, 1018. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.40 (d, J = 8.7 Hz, 2H, CH_aromatic), 7.26 (d, J = 15.9 Hz, 1H, β‐H), 6.84 (d, J = 8.7 Hz, 2H, CH_aromatic), 6.80 (d, J = 15.9 Hz, 1H, α‐H), 6.15 (s, 2H, CH_aromatic), 3.85 (s, 3H, OCH₃), 3.75 (s, 6H, 2xOCH₃), 3.27 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.73‐1.59 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 194.62 (> C═O), 162.06, 158.64, 153.02, 145.33, 130.07, 125.33, 124.49, 114.81, 112.30, 90.77 (C_aromatic and C_olefinic), 55.93, 55.44 (2xOCH₃), 49.14 (CH₂N_piperidine), 25.44 (CH₂CH₂N_piperidine), 24.34 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₃H₂₈NO₄ [M + H]⁺: 382.2018; found: 382.2018.

4.3.18. (E)‐3‐(4‐(Piperidin‐1‐yl)phenyl)‐1‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (DLC18). (Zhou et al. 2016)

Using the general procedure, DLC18 was prepared from 3’,4’,5’‐trimethoxyacetophenone and isolated as an orange solid. Yield: 46%, mp 106.8–107.6°C. FTIR (ATR) ν (cm⁻¹): 3019 (CH_aromatic), 2932, 2828 (CH_aliphatic), 1639 (> C═O), 1557, 1504, 1456, 1431, 1412, 1344, 1302, 1234, 1182, 1155, 1125, 1070. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.78 (d, J = 15.4 Hz, 1H, β‐H), 7.54 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.31 (d, J = 15.4 Hz, 1H, α‐H), 7.27 (s, 2H, CH_aromatic), 6.89 (d, J = 8.8 Hz, 2H, CH_aromatic), 3.94 (s, 6H, 2xOCH₃), 3.93 (s, 3H, OCH₃), 3.31 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 1.72‐1.58 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 189.37 (> C═O), 153.19, 153.07, 145.35, 142.02, 134.31, 130.26, 124.28, 117.41, 114.72, 105.93 (C_aromatic and C_olefinic), 60.94, 56.37 (2xOCH₃), 49.00 (CH₂N_piperidine), 25.42 (CH₂CH₂N_piperidine), 24.33 (CH₂CH₂CH₂N_piperidine). HRMS(+ ESI) m/z calcd for C₂₃H₂₈NO₄ [M + H]⁺: 382.2018; found: 382.2018.

4.3.19. (E)‐1‐Mesityl‐3‐(4‐(piperidin‐1‐yl)phenyl)prop‐2‐en‐1‐one (DLC19)

Using the general procedure, DLC19 was prepared from 2’,4’,6’‐trimethylacetophenone and isolated as a brown oil. Yield: 36%. FTIR (ATR) ν (cm⁻¹): 3024 (CH_aromatic), 2918, 2851 (CH_aliphatic), 1628 (> C═O), 1585, 1514, 1429, 1385, 1302, 1279, 1263, 1233, 1182, 1163,1126, 1063, 1020. ¹H NMR (600 MHz, CDCl ₃ ): δ 7.38 (d, J = 8.8 Hz, 2H, CH_aromatic), 7.09 (d, J = 16.0 Hz, 1H, β‐H), 6.87 (s, 2H, CH_aromatic), 6.84 (d, J = 8.8 Hz, 2H, CH_aromatic), 6.77 (d, J = 16.1 Hz, 1H, α‐H), 3.30 (t, J = 6.0 Hz, 4H, CH₂N_piperidine), 2.32 (s, 3H, CH₃), 2.19 (s, 6H, 2xCH₃), 1.77‐1.55 (m, 6H, CH₂CH₂N_piperidine). ¹³C NMR (150 MHz, CDCl ₃ ): δ 201.34 (> C═O), 153.25, 147.38, 137.92, 137.68, 134.08, 130.26, 128.23, 124.37, 123.69, 114.64 (C_aromatic and C_olefinic), 48.96 (CH₂N_piperidine), 25.36 (CH₂CH₂N_piperidine), 24.33 (CH₂CH₂CH₂N_piperidine), 21.12, 19.30 (2xCH₃). HRMS(+ ESI) m/z calcd for C₂₃H₂₈NO [M + H]⁺: 334.2171; found: 334.2174.

4.4. Biological Studies

4.4.1. Enzyme Assays and Inhibition Studies

The MAO‐A and MAO‐B activities were determined using 0.06 mM kynuramine and 0.3 mM benzylamine, respectively, as substrates, following the standard protocol. (Jeong et al. 2020) The absorbance was measured using a standard method described previously. (Rehuman et al. 2021) The SI of MAO‐B was calculated as follows: (IC₅₀ of MAO‐A)/(IC₅₀ of MAO‐B). The inhibitory effects of the test compounds were compared with those of standard compounds toloxatone and clorgyline for MAO‐A and safinamide and pargyline for MAO‐B. The IC₅₀ values of the lead compounds were measured at six different concentrations and of other effective compounds were at three different concentrations. Results were expressed as mean ± standard deviation (SD) of triplicate or duplicate experiments. The AChE and BuChE inhibitory activities of the compounds were measured using the standard protocol described previously, and donepezil was used as the reference AChE/BuChE inhibitor (Jeong et al. 2020).

4.4.2. Enzyme and Inhibition Kinetics

The enzyme kinetics study of the two potent compounds (DLC9 and DLC14) were determined at five different concentrations of benzylamine (0.0375–0.60 mM) for MAO‐B (Jeong et al. 2020; Lee et al. 2016) and three different concentrations of DLC9 (28, 56, and 112 nM) and DLC14 (14, 28, and 56 nM). The enzyme inhibition patterns and K _i values were determined by comparing the LB plots and their respective secondary plots. (Rehuman et al. 2021).

4.4.3. Reversibility Studies

The reversibility studies of compounds were evaluated by following the standard protocol. (Jeong et al. 2020; Lee et al. 2016) Residual activities of A_U and A_D were analysed at a concentration of approximately 2.0 times of IC₅₀ values after 30 min of preincubation. The recovery values of the enzyme activities were compared with the reference compounds, that is, safinamide and pargyline (reversible and irreversible inhibitors, respectively) of MAO‐B. The reversibility patterns were determined by comparing the A_U and A_D of samples (Jeong et al. 2020; Lee et al. 2016).

4.5. Molecular Docking Studies

The structure of DLC6 and DLC14 were sketched, 3D‐geometry optimized and saved in .pdb format using ChemDraw 22.2.0. Further, they were converted in to AutoDock compatible .pdbqt format using MGLTool‐1.5.7. (Morris et al. 2009) Protein preparation, docking preparation and docking runs were performed as per the protocol reported earlier. (Jayaprakash et al. 2008) Details of grid centre, grid dimension, number of runs etc. are provided in supporting material (Supporting Information Table S6). Lowest energy conformer from the largest cluster from each simulation was then analysed through PLIP server (Adasme et al. 2021; Salentin et al. 2015).

4.6. Antiparkinsonian Activity in the MPTP Induced Rat Model of Parkinsonism

The animals were randomly divided into five groups (Group I–V) each containing five rats with similar age and sex distribution. The age of all animals in each group was ~3 months. Parkinsonism was induced in all the animals except animals in Group I by the administration of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) at a dose of 30 mg/kg through intraperitoneal route once daily for 5 consecutive days. Treatment (Group III‐V) was given for a period of 7 days as given below. Subsequent pharmacological tests and the corresponding observations were recorded on Day 7, Day 14, and Day 28.

Group I (Positive Control) received vehicle only (saline, I.P.) for 7 days

Group II (Negative Control) received MPTP (30 mg/kg, I.P.) once daily for 5 days and vehicle only (saline, I.P.) for next 2 days

Group III (Treatment) received MPTP (30 mg/kg, I.P.) + Selegiline (10 mg/kg, P.O.) once daily for 5 days followed by Selegiline (10 mg/kg, P.O.) once daily for 2 days

Group V (Treatment) received MPTP (30 mg/kg, I.P.) + DLC6 (10 mg/kg, P.O.) once daily for 5 days followed by DLC6 (10 mg/kg, P.O.) once daily for 2 days

Group IV (Treatment) received MPTP (30 mg/kg, I.P.) + DLC14 (10 mg/kg, P.O.) once daily for 5 days followed by DLC14 (10 mg/kg, P.O.) once daily for 2 days

After the 5 days of induction period, animals in Group II were showing characteristic symptoms such as reduced locomotor activity, tremors, rigidity, and postural imbalance.(Yue et al. 2021) All the studies, including the Open Field Test, Pole Test, Bar Test, Rotarod Test, and Swim Test were performed on the Day 7, Day 14, and Day 28 to assess motor performance and antiparkinsonian activity of the compounds.

4.6.1. Open Field Test

A wooden open‐field box of 100 × 100 × 40 cm dimension with the floor divided into 25 equal squares of 20 × 20 cm dimension was used. Middle nine squares were marked as the centre zone and rest sixteen squares as peripheral zone. The entire area was evenly lit with the light source having ~100 lux. Each rat was placed gently at the centre of the box and allowed to move freely for 5 min. During this time, the following behaviours were recorded: (i) line crossings, (ii) rearing, (iii) entries into the centre zone, (iv) time spent in the centre (duration in seconds), (v) grooming, and the (vi) freezing or immobility (duration in seconds). These observations were recorded for Days 7, 14, and 28 after treatment. All observations were made by an observer unaware of the group assignments. Data are presented as Mean ± SD and analysed using one‐way ANOVA followed by Tukey's post‐hoc test, with p < 0.05 considered statistically significant (Rehman et al. 2022).

4.6.2. Bar Test

Each rat was gently placed with its front paws on a horizontal bar, about 1 cm thick and 9 cm above the table. The time taken to move or remove one paw from the bar was recorded in seconds as the catalepsy time. A maximum limit of 180 s was set to prevent stress. Each rat was tested three times, and the average time was used for analysis. These observations were recorded for Days 7, 14, and 28 after treatment. Results were expressed as mean ± SD and analysed using one‐way ANOVA followed by Tukey's post‐hoc test, with p < 0.05 considered significant (Ho et al. 2014).

4.6.3. Swim Test

Each rat was placed in a 90 × 45 × 45 cm tank filled with clean water to a depth of approximately 30 cm, allowing them to swim freely without touching the bottom. The water was kept at 25 ± 2°C. Every rat was given freedom to swim, and the swim time (measured in seconds) was noted as the amount of time the animal was balanced and active without sinking. A rat was taken out, dried with a towel, and kept warm if it stopped swimming or showed signs of exhaustion for longer than 10 seconds. To maintain hygiene, the water was changed frequently. On Days 7, 14, and 28 following treatments, the test was conducted (Sengupta et al. 2011).

4.6.4. Pole Test

The Pole Test was used to measure bradykinesia and motor coordination in rats. For the test, a wooden pole 50 cm high and 1 cm thick was used. The time to turn downhill (T‐turn) and the time to reach the base (T‐total) were measured twice while each rat was positioned head‐up on top of the pole. Three trials were given to each rat, and the average result was analysed. To avoid tiredness, a 20‐second cutoff time was established. In between trials, 70% ethanol was used to clean the pole. On Days 7, 14, and 28 following therapy, the test was administered. Shorter T‐turn and T‐total times showed improved motor coordination, while longer times indicated bradykinesia and motor impairment (Rehman et al. 2022).

4.6.5. Rotarod Test

Rats’ motor coordination, balance, and muscle strength were evaluated using the Rotarod Test. A revolving rod, 30 cm in length and 3 cm in diameter, was used in the setup, and it was divided into sections to test several rats simultaneously. The rod rotated at a constant pace of 20 rpm All rats were taught for 3 days to become acquainted with the equipment prior to testing. Each rat was put on the rotating rod during the test, and the amount of time it stayed on the rod without falling, as well as the fall latency time (measured in seconds), was noted. Three trials were conducted on each rat, and the average time was utilized for analysis. On Days 7, 14, and 28 following treatments, the test was conducted (Shahid Nadeem et al. 2023).

Conflicts of Interest

The authors declare no conflict of interest.

Supporting information

Supporting File

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.