KR102545193B1 - Composition for preventing or treating neurodegenerative disease comprising chalcone derivatives - Google Patents

Abstract

The present invention provides a chalcone derivative compound useful as an MAO-B inhibitor and an AChE inhibitor, a compound selected from a pharmaceutically acceptable salt thereof, a hydrate thereof or a stereoisomer thereof, and a compound containing the compound as an active ingredient to prevent or alleviate degenerative cranial nerve disease. Or, as it relates to a pharmaceutical composition for treatment, the chalcone derivative compound according to the present invention has an excellent inhibitory effect on MAO-B and AChE, so it can be usefully used for preventing, alleviating or treating degenerative cranial nerve diseases.

Description

Composition for preventing or treating neurodegenerative disease comprising chalcone derivatives

The present invention provides a chalcone derivative compound useful as an MAO-B inhibitor and an AChE inhibitor, a compound selected from a pharmaceutically acceptable salt thereof, a hydrate thereof or a stereoisomer thereof, and a compound containing the compound as an active ingredient to prevent or alleviate degenerative cranial nerve disease. or to therapeutic pharmaceutical or food compositions.

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Monoamine oxidase (MAO, EC 1.4.3.4) catalyzes the oxidative deamination of the pharmacologically important monoamine neurotransmitter and contains two MAO isoforms (MAO-A and MAO-A) in the mitochondrial outer membrane of all tissues. B) exists [1]. Because MAO-A and MAO-B have different substrate specificities, MAO-A is primarily targeted to treat depression and anxiety, while MAO-B is targeted to treat Alzheimer's disease (AD) and Parkinson's disease (PD) [2 ]. In addition, MAO is critically associated with amyloid plaque formation in Alzheimer's disease (AD) and MAO-B is expressed at high levels in the brains of Alzheimer's disease patients together with γ-secretase [3].

On the other hand, acetylcholinesterase (AChE, EC 3.1.1.7) catalyzes the degradation of acetylcholine (ACh); It is a neurotransmitter found in synapses in the cerebral cortex that is commonly known to be deficient in AD [4]. Thus, AChE inhibitors increase the level of synaptic ACh and enhance cholinergic transmission in the brain [5]. Recently, multiple targeting treatment strategies targeting MAO-B and AChE have been devised [6] and MAO and AChE inhibitors have been shown to improve cognitive function and alleviate symptoms of AD by increasing the levels of monoamines and choline esters. reported [7]. In addition, selective MAO-B inhibitors are being studied in various scaffolds [8].

Xanthoangelol and 4-hydroxyderricin are prenylated chalcones present in Angelica keiskei K., and have recently inhibited monoamine oxidase activity. and it has been demonstrated that 4-hydroxyderricin selectively inhibits MAO-B [9]. These findings suggest that chalcones have potential in the treatment of conditions associated with MAO, such as depression and AD.

Accordingly, the inventors of the present invention, in treating degenerative cranial nerve diseases such as Alzheimer's disease, studied to overcome the disadvantages of conventional treatment strategies using single-target drug molecules and to develop multi-target drug molecules that can maintain their effects even during long-term use. During the study, it was discovered that the chalcone derivative compound not only has a selective, reversible and competitive inhibitory activity against MAO-B, but also an inhibitory activity against AChE, and the present invention was completed.

Meanwhile, the papers cited in this specification are as follows.

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One object of the present invention is to provide a chalcone derivative compound having a selective and reversible inhibitory activity against monoamine oxidase-B (MAO-B) as well as an inhibitory activity against acetylcholinesterase (AChE), a pharmaceutically acceptable salt thereof, It is to provide a pharmaceutical composition for preventing or treating degenerative cranial nerve disease comprising a compound selected from hydrates thereof and stereoisomers thereof as an active ingredient.

Another object of the present invention is to provide a chalcone derivative compound having a selective and reversible inhibitory activity against MAO-B (monoamine oxidase-B) as well as an inhibitory activity against AChE (acetylcholinesterase), and a pharmaceutically acceptable salt thereof , It is to provide a food composition for preventing or improving degenerative cranial nerve disease comprising a compound selected from hydrates and stereoisomers thereof as an active ingredient.

In one aspect, the present invention provides a pharmaceutical composition for preventing or treating degenerative cranial nerve disease, comprising a chalcone derivative compound, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a compound selected from stereoisomers thereof as an active ingredient.

In the present invention, the chalcone derivative compound is 2,4'-dichloro-4-dimethylaminochalcone, 4-dimethylaminochalcone, 4-chlorochalcone (4-chlorochalcone), 4-nitrochalcone (4-nitrochalcone), and 4'-chloro-4-dimethylaminochalcone (4'-chloro-4-dimethylaminochalcone) refers to a compound selected from at least one selected from the group consisting of.

The 2,4'-dichloro-4-dimethylaminochalcone (2,4'-dichloro-4-dimethylaminochalcone) has a structure shown in Formula 1 below and is represented as Compound 1 herein.

[Formula 1]

The 4-dimethylaminochalcone (4-dimethylaminochalcone) has a structure shown in Formula 2 below, and is represented as Compound 2 herein.

[Formula 2]

The 4-chlorochalcone (4-chlorochalcone) has a structure shown in Formula 3 below, and is represented as Compound 3 herein.

[Formula 3]

The 4-nitrochalcone (4-nitrochalcone) has a structure shown in Formula 4 below, and is represented as Compound 4 herein.

[Formula 4]

The 4'-chloro-4-dimethylaminochalcone (4'-chloro-4-dimethylaminochalcone) has a structure shown in Formula 5 below, and is represented as Compound 5 herein.

[Compound 5]

The chalcone derivative compound according to the present invention may be used in the form of a pharmaceutically acceptable salt derived from an inorganic or organic acid, and preferred salts include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, glycolic acid, lactic acid, and pyruvic acid. , malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, It may be one or more selected from the group consisting of methanesulfonic acid, benzenesulfonic acid and toluenesulfonic acid.

The chalcone derivative compound or a pharmaceutically acceptable salt thereof according to the present invention may include hydrates and solvates. The hydrate may mean formed by combining the compounds of Chemical Formulas 1 to 5 with water molecules.

In the present invention, the term "stereoisomer" refers to a compound having the same molecular formula and method of connecting elements, but different spatial arrangements between atoms. The stereoisomers may be diastereomers or enantiomers. Enantiomers refer to isomers that do not overlap with their mirror images, such as the relationship between left and right hands, and are also called optical isomers. Enantiomers are classified as R (Rectus: clockwise) and S (sinister: counterclockwise) when four or more substituents on the chiral central carbon differ from each other. Diastereomers are stereoisomers that are not enantiomers, and can be divided into cis-trans isomers that arise from differences in the arrangement of atoms in space.

As used herein, the term "degenerative cranial nerve disease" refers to a disease in which degenerative changes appear in nerve cells of the central nervous system and cause various symptoms, and cognitive function, learning or memory are impaired, or accompanied by a neuroinflammatory reaction. including cranial nerve disease. Representative degenerative cranial nerve diseases according to the present invention include dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease -Jakob disease (CJD), stroke, multiple sclerosis, learning disabilities, memory impairment, etc.

The degenerative cranial nerve disease causes memory and cognition by reducing the amount of acetylcholine, a neurotransmitter, along with the formation of amyloid plaques and nerve fiber tangles containing beta-amyloid (Aβ) in brain tissue. Functional decline has been reported. Accordingly, studies to treat degenerative cranial nerve diseases such as Alzheimer's disease by inducing inhibition of acetylcholine degraders such as acetylcholinesterase (AChE) have been attempted.

In addition, degenerative cranial nerve diseases are caused by oxidative stress caused by hydrogen peroxide due to the increase in the activity of 'MAO-B', an enzyme that oxidatively deaminates neurotransmitters such as dopamine, serotonin, adrenaline, and noradrenaline, and non-biocomponent amines. induced by oxidative stress, MAO-B inhibitors can reduce the formation of oxygen radicals and increase the amount of available monoamines in the brain. In cranial nerve disease and brain injury, MAO-B promotes putrescine metabolism in reactive astrocytes, leading to excessive production of GABA. Therefore, MAO-B inhibitors act as inhibitors of GABA production by astrocytes and have the effect of restoring nerve signal transmission and brain function.

The pharmaceutical composition according to the present invention has excellent activity in inhibiting monoamine oxidase-B (MAO-B) and acetylcholinesterase (AChE). Therefore, since the pharmaceutical composition of the present invention has excellent MAO-B and AChE inhibitory activity, it can be used for the purpose of treatment, prevention, and alleviation of degenerative cranial nerve diseases.

In the present invention, the inhibition of MAO-B may be inhibition of MAO-B activity or inhibition of MAO-B biosynthesis. The inhibition of MAO-B activity may be a reversible inhibition, and may have a selectivity of several times or more, for example, 10 times or more, for MAO-B than for MAO-A. Preferably, the chalcone derivative compound represented by Chemical Formulas 1 to 5, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a stereoisomer thereof can inhibit MAO-B activity reversibly, selectively and competitively.

In one specific embodiment, as a result of evaluating the inhibitory activity of 12 natural or synthetic chalcone derivative compounds against MAO-B and AChE, the chalcone derivative compounds of formulas 1 to 5 according to the present invention showed significant inhibition against MAO-A. It was confirmed that, while showing no activity, it showed excellent inhibitory efficacy selectively against MAO-B as well as excellent inhibitory efficacy against AChE. In particular, it was confirmed that the chalcone derivative compounds of Chemical Formulas 1 to 5, preferably the chalcone derivative compounds of Chemical Formula 2 according to the present invention are useful as MAO-B/AChE dual targeting inhibitors capable of treating degenerative cranial nerve diseases such as Alzheimer's disease. .

As described above, the chalcone derivative compounds of Chemical Formulas 1 to 5 according to the present invention are selective MAO-B activity inhibitors and have high inhibitory activity against the MAO-B enzyme as well as high inhibitory activity against AChE, thereby preventing degenerative It was proved that it can be used as a composition for treating cranial nerve diseases.

The pharmaceutical composition contains 0.001 to 50% by weight of at least one compound selected from among chalcone derivative compounds represented by Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof, and stereoisomers thereof, based on the weight of the total composition. %, preferably 0.001 to 20% by weight, more preferably 0.001 to 10% by weight.

The pharmaceutical composition may be applied to lab animals such as, but not limited to, mice, rabbits, rats, guinea pigs, or hamsters, or primates including humans, preferably to primates including humans, and more preferably to humans.

As used herein, 'treatment' refers to alleviation or improvement of symptoms, reduction of extent of disease, delay or alleviation of disease progression, improvement of disease state, alleviation or stabilization, partial or complete recovery, prolongation of survival, and other beneficial treatment results. It can be used in an all-inclusive sense.

Depending on the usage mode and method of the pharmaceutical composition of the present invention, the content of the active ingredient, the chalcone derivative compound represented by Formulas 1 to 5, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a stereoisomer thereof, may be determined by a person skilled in the art. It can be used by adjusting it appropriately according to.

A compound selected from the chalcone derivative compounds of Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof, and stereoisomers thereof may be included alone in the pharmaceutical composition, or other pharmacologically acceptable carriers, excipients, It may also be included with diluents or auxiliary ingredients.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , dextrin, calcium carbonate, propylene glycol, liquid paraffin, and at least one selected from the group consisting of physiological saline, but is not limited thereto, and all conventional carriers, excipients, or diluents may be used. In addition, the pharmaceutical composition may include conventional fillers, extenders, binders, disintegrants, anti-coagulants, lubricants, wetting agents, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective chloride, glycerin , A flavoring agent, an emulsifier or a preservative may be further included.

The compounds selected from the chalcone derivative compounds of Chemical Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof and stereoisomers thereof according to the present invention, together with other MAO-B inhibitors or AChE inhibitors for treating degenerative cranial nerve diseases The therapeutic effect of an MAO-B selective inhibitor and an AChE inhibitor can be enhanced by co-administration.

Specifically, the pharmaceutical composition further includes one or more other MAO-B inhibitors or AChE inhibitors known to be effective for the treatment or prevention of degenerative cranial nerve diseases in addition to the active ingredient, and can be used as a combination therapy applied simultaneously or at different times. . Other MAO-B inhibitors that can be applied in the combination therapy include, for example, the group consisting of selegiline, rasagiline, safinamide, lazabemide, and pargyline. It may include one or more compounds selected from, but is not limited thereto. In addition, other AChE inhibitors may include, for example, at least one compound selected from the group consisting of tacrine, donepezil, galantamine, and rivastigmine. It is not limited to this.

The method of administering the pharmaceutical composition can be either oral or parenteral, and for example, it can be administered through various routes including oral, transdermal, subcutaneous, intravenous, intramuscular or intracerebroventricular injection. In addition, the formulation of the composition may vary depending on the method of use, and is formulated using a method well known in the art to which the present invention pertains so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be. In general, solid preparations for oral administration include tablets (TABLETS), pills, soft or hard capsules (CAPSULES), pills (PILLS), powders (POWDERS) and granules (GRANULES), etc., and these preparations include one or more Excipients, for example, may be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, Sweetening agents, flavoring agents, preservatives, and the like may be included. Forms for parenteral administration include creams, lotions, ONITMENTS, PLASTERS, LIQUIDS AND SOLUTIONS, aerosols, FRUIDEXTRACTS, and elixirs. It may be in the form of (ELIXIR), INFUSIONS, SACHET, PATCH, or INJECTIONS, and may be in the form of an isotonic aqueous solution or suspension preferably in the case of an injectable formulation. .

The pharmaceutical composition may further contain adjuvants such as sterilizers, preservatives, stabilizers, hydration agents or emulsion accelerators, salts and/or buffers for osmotic pressure control, and other therapeutically useful substances, and conventional mixing and granulation Alternatively, it may be formulated according to a coating method, and in addition, it may be formulated using an appropriate method known in the art.

In addition, the dosage of the pharmaceutical composition can be determined in consideration of the administration method, the age and sex of the user, the severity of the patient, the condition, the absorption of the active ingredient in the body, the inactivity rate, and the drugs used in combination, once or several times. It can be administered in divided doses.

In another aspect, the present invention is directed to administering a therapeutically effective amount of a compound selected from at least one chalcone derivative compound among the compounds represented by Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof, and stereoisomers thereof. It provides a method for preventing or treating degenerative cranial nerve disease, comprising the step.

Preferably, the treatment method may further include identifying a patient in need of prevention or treatment of the degenerative cranial nerve disease prior to the administration step.

The "therapeutically effective amount" of the present invention means an amount of an active ingredient effective for preventing or treating a degenerative cranial nerve disease, for mammals, and the therapeutically effective amount is the type of disease, the severity of the disease, the active ingredient contained in the composition, and Adjusted according to various factors including the type and content of other components, type of dosage form and patient's age, weight, general health condition, gender and diet, administration time, route of administration and blood clearance of the composition, duration of treatment, drugs used concurrently It can be.

According to the present invention, a compound selected from at least one chalcone derivative compound, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a stereoisomer thereof among the compounds represented by Formulas 1 to 5 not only inhibits AChE but also reversibly inhibits MAO-B. As a result, by inhibiting selectively and competitively, degenerative cranial nerve diseases such as Alzheimer's disease can be prevented or treated efficiently and with few side effects.

In another aspect, the present invention relates to a degenerative cranial nerve comprising a compound selected from at least one chalcone derivative compound among the compounds represented by Chemical Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof, and stereoisomers thereof. It provides a food composition for preventing or improving diseases.

The food composition of the present invention may include all food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food and health functional food.

The health functional food is a food that emphasizes the bioregulatory function of food, and is a food that has added value to act and express for a specific purpose using physical, biochemical, and bioengineering methods. The ingredients of these health functional foods are designed and processed to sufficiently exert the body's control functions related to body defense and regulation of body rhythm, prevention and recovery of diseases, and are food additives, sweeteners, or functional foods acceptable as food. may contain raw materials.

When a compound selected from the chalcone derivative compounds of Chemical Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof, and stereoisomers thereof is used as a health functional food (or health functional beverage additive), the compound is added as it is or It can be used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the compound may be appropriately determined depending on the purpose of its use (prevention, health or improvement, therapeutic treatment).

The health functional food is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protective colloidal thickeners agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain fruit flesh for preparing fruit and vegetable beverages. These components may be used alone or in combination, and the proportion of these additives is generally selected from the range of 0.001 to 50 parts by weight per total weight of the composition.

There is no particular limitation on the type of health functional food. Foods to which the compound may be added include sausages, meats, breads, chocolates, snacks, candies, confectionery, ramen, pizza, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, alcohol Beverages and vitamin complexes. When formulated into a beverage, the liquid component added in addition to the novel lactic acid bacteria is not limited thereto, but may contain various flavoring agents or natural carbohydrates as additional components, as in conventional beverages. The aforementioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.) and polysaccharides (eg, common sugars such as dextrins, cyclodextrins, etc.), and xylitol, sorbitol. , sugar alcohols such as erythritol.

The food composition contains 0.001 to 50% by weight of at least one compound selected from among the chalcone derivative compounds represented by Formulas 1 to 5, pharmaceutically acceptable salts thereof, hydrates thereof, and stereoisomers thereof, based on the weight of the total composition. %, preferably 0.001 to 20% by weight, more preferably 0.001 to 10% by weight.

The chalcone derivative compounds represented by Chemical Formulas 1 to 5 according to the present invention not only play an important role in dopamine metabolism in the brain, but also have excellent inhibitory effects on MAO-B, which is known to inhibit brain nerve cell damage, and Since the effect of inhibiting the degradation of acetylcholine, a transmitter, that is, the activity of AChE is also excellent, it can be usefully used for the purpose of treatment, prevention, and alleviation of degenerative cranial nerve diseases.

1 is a diagram showing the degree of recovery after dialysis of MAO-B by compound 2 according to an embodiment of the present invention. Razabemide and pargyline were used as reference reversible and irreversible MAO-B inhibitors, respectively. The concentrations of inhibitors used were 0.060 μM for compound 2; Razabemide at 0.065 μM; and pargylin is 0.20 μM. Results are the average of duplicate experiments.
2 is a diagram showing a Lineweaver-Burk plot of MAO-B inhibition by compound 2 according to an embodiment of the present invention (A) and a second-order plot of the slope of the Lineweaver-Burk plot for compound 2 (B) am. Substrate concentrations ranged from 0.03 to 0.6 mM. Experiments were performed at three inhibitor concentrations, ˜0.5, 1.0 and 2.0 fold IC ₅₀ values. The initial rate was expressed as an increase in absorbance per minute.
3 and 4 are drawings showing docking simulation results of compounds 1 to 12 for MAO-A according to an embodiment of the present invention. Here, A is compound 1 (2,4'-dichloro-4-dimethylaminochalcone); B is compound 2 (4-dimethylaminochalcone); C is compound 3 (4-chlorochalcone); D is compound 4 (4-nitrochalcone); E is compound 5 (4'-chloro-4-dimethylaminochalcone); F is compound 6 (4-carboxymethylchalcone); G is compound 7 (sappanchalcone); H is compound 8 (3-deoxysappanchalcone); I is compound 9 (broussochalcone A); J is compound 10 (4-hydroxyderricin); K is compound 11 (xanthoangelol); and L is compound 12 (2,2'-dihydroxy-4',6'-dimethoxychalcone).
5 and 6 are drawings showing docking simulation results of compounds 1 to 12 for MAO-B according to an embodiment of the present invention. Here, A is compound 1 (2,4'-dichloro-4-dimethylaminochalcone); B is compound 2 (4-dimethylaminochalcone); C is compound 3 (4-chlorochalcone); D is compound 4 (4-nitrochalcone); E is compound 5 (4'-chloro-4-dimethylaminochalcone); F is compound 6 (4-carboxymethylchalcone); G is compound 7 (sappanchalcone); H is compound 8 (3-deoxysappanchalcone); I is compound 9 (broussochalcone A); J is compound 10 (4-hydroxyderricin); K is compound 11 (xanthoangelol); and L is compound 12 (2,2'-dihydroxy-4',6'-dimethoxychalcone).
7 and 8 are drawings showing docking simulation results of compounds 1 to 12 for AchE according to an embodiment of the present invention. Here, A is compound 1 (2,4'-dichloro-4-dimethylaminochalcone); B is compound 2 (4-dimethylaminochalcone); C is compound 3 (4-chlorochalcone); D is compound 4 (4-nitrochalcone); E is compound 5 (4'-chloro-4-dimethylaminochalcone); F is compound 6 (4-carboxymethylchalcone); G is compound 7 (sappanchalcone); H is compound 8 (3-deoxysappanchalcone); I is compound 9 (broussochalcone A); J is compound 10 (4-hydroxyderricin); K is compound 11 (xanthoangelol); and L is compound 12 (2,2'-dihydroxy-4',6'-dimethoxychalcone).

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

1. Experimental Materials and Methods

1-1. Experiment preparation

The medicinal plants Caesalpinia sappan L., Broussonetia papyrifera L., Angelica keiskei , and purslane ( Portulaca oleracea L.) were purchased from the Korean herbal market between 2010 and 2015. Organic solvents, namely methanol (MeOH), n-hexane (Hx), ethyl acetate (EtOAc), dichloromethane (CH ₂ Cl ₂ ), chloroform (CHCl ₃ ) and butanol (BuOH) were purchased from Duksan Chemical Co., Ltd. . Column chromatography using silica gel 60 (Merck 70-230 mesh, ASTM, Germany), ODS-A (12 nm, S-150 m, YMC, Tokyo, Japan) and Sephadex LH-20 gel (GE Healthcare, Sweden) was performed. NMR spectra were recorded on a JEOL ECX-500 spectrometer (JEOL Ltd, Japan) at 500 MHz for ¹ H and 125 MHz for ¹³ C.

1-2. Extraction and isolation of active compounds

Dried heartwood (15.0 kg) of Caesalpinia sappan L. was extracted with MeOH, and the extract (1.5 kg) was partitioned between H ₂ O and EtOAc. The EtOAc soluble fraction (960.0 g) was separated into 14 fractions (CSE 1 to 14) by silica gel column chromatography using a CHCl ₃ /MeOH gradient (50:1 to 0:1). Fraction CSE 5 was purified by preparative HPLC (Spot II, Armen, France) using a Watchers 120 ODS-AP column at a flow rate of 10 ml/min with 45% MeOH as eluent to give two fractions (CSE 5-1 ~ CSE 5-2). Fraction CSE 5-2 was subjected to Sephadex LH-20 column chromatography (MeOH: H ₂ O = 1: 1) to obtain sappanchalcone (7, 52.0 mg). Fraction CSE 9 was subjected to ODS-A column chromatography using a MeOH/H ₂ O gradient (30: 1 to 1: 0) to obtain 7 fractions (CSE 9-1 to CSE 9-7). Fraction CSE 9-1 was purified by preparative HPLC using a YMC packed ODS-A column with 70% MeOH as eluent at a flow rate of 5 ml/min to obtain 3-deoxysappanchalcone (8, 51.0 mg) was obtained.

에서 5시간 동안 MeOH로 추출하고, 그 획득한 추출물(230.0 g)을 2,000 ml의 H₂O 및 동일한 부피의 EtOAc로 분배하였다. EtOAc 가용성 분획(85.0 g)을 Hx/EtOAc 구배 (100: 0 내지 80: 20)를 사용하여 실리카겔 컬럼 크로마토그래피에 의해 9개의 분획(BPE 1-9)으로 분리하였다. 분획 BPE 4를 100 % MeOH을 용리액으로 사용하는 세파덱스 LH-20 컬럼 크로마토그래피로 분리하여 브로우소칼콘 A (broussochalcone A) (9, 100.0 mg)를 수득하였다.Dried roots (5.0 kg) of Broussonetia papyrifera L.

was extracted with MeOH for 5 h, and the obtained extract (230.0 g) was partitioned between 2,000 ml of H ₂ O and an equal volume of EtOAc. The EtOAc soluble fraction (85.0 g) was separated into 9 fractions (BPE 1-9) by silica gel column chromatography using a Hx/EtOAc gradient (100:0 to 80:20). Fraction BPE 4 was separated by Sephadex LH-20 column chromatography using 100% MeOH as an eluent to obtain broussochalcone A (9, 100.0 mg).

Angelica keiskei dried leaves (20 kg) were extracted with MeOH for 24 hours at room temperature. The MeOH extract (2.1 kg) was partitioned with 5,000 ml of H ₂ O and an equal volume of Hx. The Hx soluble fraction (160 g) was subjected to silica gel column chromatography using a CH ₂ Cl ₂ /MeOH gradient (99.9: 0.1 to 80: 20) to give 13 fractions (AKH 1-13). Fraction AKH 9 (3.65 g) was subjected to ODS-A column chromatography (MeOH: H ₂ O = 10: 1) to obtain 4-hydroxyderricin (10, 300 mg), and fraction AKH 12 (8.68 g) was subjected to silica gel column chromatography using a Hx/EtOAc gradient (98:2 to 50:50) to give xanthoangelol (11,1,500 mg).

Dried purslane ( Portulaca oleracea L.) (9.67 kg) was extracted with CH ₂ Cl ₂ at room temperature for 24 h, and the obtained extract (1.2 kg) was partitioned with 3,000 ml of 85% aqueous MeOH and an equal volume of Hx. did The 85% MeOH soluble fraction (120.45 g) was subjected to ODS-A gel column chromatography using a MeOH/H ₂ O gradient (1: 1 to 1: 0) to give 7 fractions (POM 1-7). . POM 7 (12.06 g) was subjected to silica gel column chromatography using an n-hexane/EtOAc gradient (1: 0 to 0: 1) to obtain 18 fractions (POM 7-1 to POM 7-18). POM 7-2 (3.93 g) was subjected to preparative HPLC using a YMC packed ODS-A column and eluted with 75% MeOH to form 2,2'-dihydroxy-4',6'-dimethoxychalcone (2 ,2'-dihydroxy-4',6'-dimethoxychalcone) (12, 100 mg) was obtained.

1-3. compounds and enzymes

Recombinant human MAO-A and MAO-B, kynuramine, benzylamine, toloxatone, lazabemide, acetylcholinesterase (AChE, Electrophorus electricus), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide , ACTI) and tacrine were purchased from Sigma-Aldrich (St. Louis, MO, USA) [10]. Reference irreversible inhibitors, clorgyline and pargyline, were purchased from BioAssay Systems (Hayward, CA, USA) [11]. Six synthetic chalcone derivatives were purchased from Sigma-Aldrich. All other chemicals used were of reagent grade.

1-4. enzyme assay

MAO-A and -B activities were measured using continuous spectrophotometry as previously described [12, 13]. The K _m values of kinuramine for MAO-A and benzylamine for MAO-B were 0.040 and 0.13 mM, respectively. In this experiment, the concentrations of kinuramine (0.06 mM) and benzylamine (0.3 mM) were 1.5 Х K _m and 2.3 Х K _m values, respectively. AChE activity was assayed [10, 15] with minor modifications to the method developed by Ellman et al. [14]. The reaction was continuously monitored at 412 nm for 10 min using ~0.2 U/mL of AChE in the presence of 0.5 mM DTNB and 0.5 mM ACTI in 0.5 ml reaction mixture. The inhibitory activity was measured after pre-incubating the enzyme with the inhibitor for 15 minutes before adding DTNB and ATCI. Response rate was expressed as change in absorbance per minute (min).

1-5. Inhibitory activity and enzyme kinetics

Inhibition of MAO-A, MAO-B and AChE by chalcone was initially assayed at a chalcone concentration of 10 μM. The IC ₅₀ values of chalcone and reference reversible inhibitors (toloxatone, razabemide and tacrine against MAO-A, MAO-B and AChE) were determined. As described in the prior literature [16], the time-dependence, kinetic parameters, inhibition type and _Ki value of the most potent MAO-B inhibitor (Compound 2) were investigated. The kinetics of MAO-B inhibition by compound 2 were analyzed in the presence or absence of each inhibitor at concentrations of five different substrate concentrations (0.03, 0.06, 0.15, 0.3 or 0.6 mM) and their IC ₅₀ values of ~0.5 Х, 1.0 Х and 2.0 Х was investigated in the absence [10]. Inhibition patterns and _Ki values were determined using a Lineweaver-Burk plot and a quadratic plot of the slope of the plot.

1-6. Inhibitor reversibility assay

The reversibility of MAO-B inhibition by compound 2 was demonstrated at concentrations of ~2 Х IC ₅₀ values, namely 0.060 μM for compound 2, 0.065 μM for razabemide and 0.20 μM for pargyline, as described in [11]. were investigated by dialysis at μM. After pre-incubation of the chalcone or reference inhibitor with MAO-B for 30 minutes, residual activity was measured for non-dialysis and dialyzed samples. Relative values for activity without dialysis (AU) and activity with dialysis (AD) were then calculated and compared to respective controls without inhibitor. Reversibility was determined by comparing the AU and AD values of the inhibitor to those of the reference inhibitor.

1-7. Docking simulations of six synthetic chalcones to MAO-A, MAO-B and AchE

Autodock Vina [17] with automatic docking was used to simulate the docking of six chalcones to the MAO enzyme or AchE. To define the docking sites for MAO-A, MAO-B and AChE, the MAO-A/7-methoxy-1-methyl-9H-beta-carboline complex (MAO-A/7-methoxy-1-methyl- 9H-beta-carboline complex) (PDB ID: 2Z5X), MAO-B/pioglitazone complex (PDB ID: 4A79) and AChE/3-[(1S)-1-(dimethylamino) A series of known active sites previously defined from the ethyl]phenol (SAF) complex (PDB ID: 1GQS) was used. To prepare the docking simulation, we performed three steps described in literature [13, 15]. 1) Energy minimization using ChemOffice program (http://www.cambridgesoft.com); 2) docking simulation using Chimera [18]; 3) Checking possible hydrogen bond interactions using the Chimera program [19].

2. Results

2-1. Isolation and identification of compounds

Six natural chalcone derivatives were isolated and identified by comparing NMR data with literature values; Saphanchalcone (Compound 7) [20,21], 3-Deoxysaphanchalcone (Compound 8) [20,21], Brousochalcon A (Compound 9) [22], 4-Hydroxydericine (Compound 10) [23], xanthoangerol (compound 11) [24], and 2,2'-dihydroxy-4',6'-dimethoxychalcone (compound 12) [25]. Six synthetic chalcone derivatives were selected in view of their known ability to inhibit MAO enzymes. The structures of the 12 chalcones are detailed in Table 1 below.

Compound 1 (2,4'-dichloro-4-dimethylaminochalcone) Compound 2 (4-dimethylaminochalcone)

Compound 3 (4-chlorochalcone) Compound 4 (4-nitrochalcone)

Compound 5 (4'-chloro-4-dimethylaminochalcone) Compound 6 (4-carboxymethylchalcone)

Compound 7 (sappanchalcone) Compound 8 (3-deoxysappanchalcone)

Compound 9 (broussochalcone A) Compound 10 (4-hydroxyderricin)

Compound 11 (xanthoangelol) Compound 12 (2,2'-dihydroxy-4',6'-dimethoxychalcone)

2-2. Assay of inhibitor activity

The MAO-A, MAO-B and AChE inhibitory activities of 12 chalcones were tested at a concentration of 10 μM. At 10 μM, most of the 12 compounds inhibited MAO-B by more than 50%, except for compounds 6 and 11 (Table 1), and most of the synthetic chalcones (compounds 1–5) inhibited MAO-B from natural chalcones (compounds 7-12) inhibited MAO-B more. Compound 2 most potently inhibited MAO-B with an IC ₅₀ value of 0.029 μM, followed by compounds 5, 4, 1, and 3 with IC ₅₀ values of 0.061, 0.066, 0.075, and 0.082 μM, respectively. was inhibited (Table 1). Compound 6 was a much weaker inhibitor (IC ₅₀ = 10.6 μM) than other synthetic chalcones. For the six native chalcones, compound 8 most strongly inhibited MAO-B (IC ₅₀ = 0.38 μM), and prenylated chalcone 10 effectively inhibited MAO-B (IC ₅₀ = 1.39 μM), while the other 2 The two prenylated chalcones (9 and 11) were relatively inefficient (IC ₅₀ values of 7.02 and 13.2 μM, respectively).

From the results of comparing the structures of the chalcone derivatives in this experiment, it was found that the functional group of C-4 affects the activity of MAO-B. The presence of a dimethylamino group in compound 1 increased MAO-B inhibition, whereas introduction of a carboxymethyl group at the same position (compound 6) significantly reduced MAO-B inhibition. In addition, the presence of substituents at C-2 and C-4' reduced MAO-B inhibition, and chlorine substitution of hydrogen at these positions reduced MAO-B inhibition. In this example, it can be seen that compounds 1 and 5 are weaker inhibitors than compound 2.

Chalcone derivatives also effectively inhibited MAO-A, but their inhibitory effect on MAO-A was weaker than that on MAO-B (Table 1). Compounds 1, 2, 9, and 12 effectively inhibited MAO-A with IC ₅₀ values of 0.18, 3.28, 0.72, and 0.39 μM, respectively, while the remaining 8 had IC ₅₀ values greater than 10 μM. The selectivity index (SI) values of compounds 5, 4, 3 and 2 for MAO-A versus MAO-B were 201.6, 137.9, 121.3 and 113.1, whereas the value for compound 1 (2.4) was substantially lower. (Table 2).

The substitution at C-2 was considered important for MAO-A inhibition. Compound 1, in which C-2 hydrogen was substituted with chlorine, inhibited MAO-A much more than Compound 5. In addition, substitution of the chlorine atom at C-4' in compound 5 reduced the inhibitory activity compared to compound 2. The isoprenyl group located at C-5' appeared to be important as compound 9 inhibited MAO-A much more than compounds 10 and 11. However, compared to compound 8, since compound 7 showed weaker inhibitory activity, the hydroxyl group at the C-3 position reduced the inhibitory activity of MAO-A.

We also investigated the ability of 12 chalcones to inhibit AChE, a therapeutic target for AD. Compound 4 effectively inhibited AChE with an IC ₅₀ value of 1.25 μM, followed by compounds 11, 1, 3, 9, 5 and 2 (IC ₅₀ values of 1.97, 2.46, 2.79, 5,63, 6.02 and 6.07, respectively). μM) inhibited AChE. The other 6 showed weak inhibitory activity (IC ₅₀ values > 10 μM). Accordingly, it was found that Compound 2 is a selective inhibitor of MAO-B and AChE.

2-3. Reversibility assay of MAO-B inhibition

MAO-B activity was not reduced when preincubated with compound 2 for up to 30 min, indicating that the interaction between the two was instantaneous.

The reversibility of MAO-B inhibition by compound 2 was investigated by dialysis. Inhibition of MAO-B by compound 2 was found to recover from 30.5% (AU) to 92.5% (AD), similar to that investigated for razabemide (reversible inhibitor; 28.3 to 86.0%), but also 1), no recovery was observed with pargyline (irreversible inhibitor; 29.9 to 28.6%). These results indicate that compound 2 reversibly inhibits MAO-B.

2-4. Analysis of inhibition patterns

The mode of MAO-B inhibition by compound 2 was investigated using a Lineweaver-Burk plot. The MAO-B inhibition plot by compound 2 was linear and crossed the y axis (Fig. 2(A)). A quadratic plot of the slope of the Lineweaver-Burk plot against the inhibitor concentration indicated that the Ki value of compound 2 for MAO-B inhibition was 0.0066 ± 0.0006 μM (Fig. 2(B)). These results indicate that compound 2 acts as a competitive inhibitor of MAO-B.

2-5. Molecular docking simulation

Docking simulations showed that six synthetic chalcones were found in the binding site of 7-methoxy-1-methyl-9H-beta-carbolin complexed with MAO-A (PDB: 2Z5X), pioglitazone complexed with MAO-B (PDB: 4A79) and 3-[(1S)-1-(dimethylamino)ethyl]phenol complexed with AChE (PDB: 1GQS). The binding affinities of all chalcones to MAO-B were greater than either MAO-A or AChE as determined by AutoDock Vina (Table 3). Correlating with the IC ₅₀ values of chalcones (Table 2), the binding energies of compound 1 to MAO-A and compound 11 to AChE are the highest, whereas the binding energies of compound 2 to MAO-B and compound 4 to AChE are were the second respectively. In addition, docking simulations showed that compounds 1 to 5 bound to MAO-B by hydrogen bonding with the Cys172 residue, and compound 6 did not form a hydrogen bond with Cys172 (FIG. 5). However, docking simulations did not predict hydrogen bond formation between chalcone and MAO-A or AChE except for compound 6, which was predicted to form a hydrogen bond with Phe288 of AChE (FIGS. 3 to 8). Parameters for calculating the experimental binding energies of chalcones tested with MAO-A, MAO-B or AChE are shown in Table 4 below. These results explain the selectivity of chalcones for MAO-B.

As a result, it was shown that the binding patterns of MAO-B and 12 chalcones were similar, and their binding affinity to MAO-B was generally greater than that to MAO-A or AChE. Although the binding scores obtained from docking simulations did not correlate perfectly with the IC ₅₀ values, compounds 1 to 5 had binding energies of >9.0 kcal/mol, indicating that they could effectively inhibit MAO-B. In addition, it was found that 5 out of 6 synthetic chalcones (Compounds 1 to 5) form a hydrogen bond with MAO-B at Cys172. This hydrogen bond can be formed by the dimethylamino group located at C-4, and the C-4 carboxymethyl of compound 6 can interfere with binding to MAO-B due to the steric factor and the possible charge of the carboxyl group at analytical pH 7.4. It was suggested that

MAO-A (kcal/mol) MAO-B (kcal/mol) AChE (kcal/mol) Docking Exp Docking Exp Docking Exp One -8.80 -9.74 -9.50 -10.43 -8.10 -8.67 2 -8.30 -8.02 -9.60 -10.99 -7.80 -8.13 3 -7.50 -7.37 -9.40 -10.37 -8.10 -8.59 4 -8.00 -7.42 -9.50 -10.50 -8.20 -9.07 5 -7.30 -7.24 -9.90 -10.52 -7.60 -8.14 6 -7.30 -6.54 -8.10 -6.71 -7.10 -7.16 7 -7.20 -6.54 -8.80 -8.89 -7.10 -7.29 8 -7.40 -7.36 -8.90 -9.47 -7.70 -7.01 9 -8.70 -8.92 -8.90 -7.74 -7.70 -8.18 10 -7.10 -6.54 -9.00 -8.70 -7.70 -7.53 11 -5.40 -6.54 -8.20 -7.36 -8.40 -8.80 12 -7.60 -9.29 -9.00 -8.30 -7.40 -7.01

Docking: docking analysis; Exp, experimental analysis

Binding energies of compounds 1 to 12 to MAO-A by experiment and docking analysis compound MAO-A [E] (μM) [S] (μM) Km (μM) IC ₅₀ (μM) Ki (μM) Temp (K) R
(kcal K ^-1 mol ^-1 ) Exp E Docking E One 0.125 60 40 0.18 0.072 298.15 1.987E-03 -9.74 -8.80 2 0.125 60 40 3.28 1.312 298.15 1.987E-03 -8.02 -8.30 3 0.125 60 40 9.95 3.980 298.15 1.987E-03 -7.37 -7.50 4 0.125 60 40 9.10 3.640 298.15 1.987E-03 -7.42 -8.00 5 0.125 60 40 12.30 4.920 298.15 1.987E-03 -7.24 -7.30 6 0.125 60 40 >40.00 16.000 298.15 1.987E-03 -6.54 -7.30 7 0.125 60 40 >40.00 16.000 298.15 1.987E-03 -6.54 -7.20 8 0.125 60 40 10.10 4.040 298.15 1.987E-03 -7.36 -7.40 9 0.125 60 40 0.72 0.288 298.15 1.987E-03 -8.92 -8.70 10 0.125 60 40 >40.00 16.000 298.15 1.987E-03 -6.54 -7.10 11 0.125 60 40 >40.00 16.000 298.15 1.987E-03 -6.54 -5.40 12 0.125 60 40 0.39 0.156 298.15 1.987E-03 -9.29 -7.60

*Molecular weight of MAO-A was used in dimer form of 120 kDa.

Binding energies of compounds 1 to 12 to MAO-B by experiment and docking analysis compound MAO-B [E] (μM) [S] (μM) Km (μM) IC ₅₀ (μM) Ki (μM) Temp (K) R (kcal K ^-1 mol ^-1 ) Exp E Docking E One 0.174 300 130 0.075 0.0227 298.15 1.987E-03 -10.43 -9.50 2 0.174 300 130 0.029 0.0088 298.15 1.987E-03 -10.99 -9.60 3 0.174 300 130 0.082 0.0248 298.15 1.987E-03 -10.37 -9.40 4 0.174 300 130 0.066 0.0200 298.15 1.987E-03 -10.50 -9.50 5 0.174 300 130 0.064 0.0193 298.15 1.987E-03 -10.52 -9.90 6 0.174 300 130 40.000 12.0930 298.15 1.987E-03 -6.71 -8.10 7 0.174 300 130 1.010 0.3053 298.15 1.987E-03 -8.89 -8.80 8 0.174 300 130 0.380 0.1149 298.15 1.987E-03 -9.47 -8.90 9 0.174 300 130 7.020 2.1223 298.15 1.987E-03 -7.74 -8.90 10 0.174 300 130 1.390 0.4202 298.15 1.987E-03 -8.70 -9.00 11 0.174 300 130 13.200 3.9907 298.15 1.987E-03 -7.36 -8.20 12 0.174 300 130 2.710 0.8193 298.15 1.987E-03 -8.30 -9.00

*The molecular weight of MAO-B was used in dimer form of 115 kDa.

Binding energies of compounds 1 to 12 to AChE by experimental and docking analysis compound AChE [E] (μM) [S] (μM) Km (μM) IC ₅₀ (μM) Ki (μM) Temp (K) R
(kcal K ^-1 mol ^-1 ) Exp E Docking E One 0.0035 500 110 2.46 0.4436 298.15 1.987E-03 -8.67 -8.10 2 0.0035 500 110 6.07 1.0946 298.15 1.987E-03 -8.13 -7.80 3 0.0035 500 110 2.79 0.5031 298.15 1.987E-03 -8.59 -8.10 4 0.0035 500 110 1.25 0.2254 298.15 1.987E-03 -9.07 -8.20 5 0.0035 500 110 6.02 1.0856 298.15 1.987E-03 -8.14 -7.60 6 0.0035 500 110 31.30 5.6443 298.15 1.987E-03 -7.16 -7.10 7 0.0035 500 110 25.01 4.5100 298.15 1.987E-03 -7.29 -7.10 8 0.0035 500 110 40.00 7.2131 298.15 1.987E-03 -7.01 -7.70 9 0.0035 500 110 5.63 1.0152 298.15 1.987E-03 -8.18 -7.70 10 0.0035 500 110 16.86 3.0403 298.15 1.987E-03 -7.53 -7.70 11 0.0035 500 110 1.97 0.3552 298.15 1.987E-03 -8.80 -8.40 12 0.0035 500 110 40.00 7.2131 298.15 1.987E-03 -7.01 -7.40

*The molecular weight of AChE was used as a tetramer of 280 kDa as described in the supplier's specifications.

3. Implications

Chalcones are open-chain flavonoids with a wide range of biological activities including anticancer, anti-inflammatory and antibacterial activity [26]. However, relatively few studies have investigated the inhibitory effect of MAO-A or MAO-B by natural chalcones. In a previous study, 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4'' from Gentiana lutea ) -O-isoprenyldihydrochalcone) and its hydrolysis products showed IC ₅₀ values of 48.7 and 6.2 μM for MAO-B, respectively, and inhibited MAO-B more strongly than MAO-A [27]. In another study, 4-hydroxyderricin, among the two prenylated chalcones found in Angelica keiskei K., potently and selectively inhibited MAO-B (IC ₅₀ was 3.43 μM), but Xanthoangelol inhibited MAO-A and MAO-B with IC ₅₀ values of 43.4 and 43.9 μM, respectively, whereas MAO-A (IC ₅₀ of 3,520 μM) did not [9]. In addition, isoliquiritigenin from Sinofranchetia chinensis inhibited MAO-A and MAO-B with IC ₅₀ values of 13.9 and 47.2 μM, respectively [28]. This experiment was performed using the rat mitochondrial MAO enzyme. However, pinostrobin chalcone from Renealmia Alpinia was reported to moderately inhibit recombinant human MAO-A and MAO-B with IC ₅₀ values of 6.3 and 10.0 μM, respectively [29]. .

Chalcone derivatives are of interest as starting points for drug design [30] and have been proposed to represent scaffolds for MAO inhibitors [31]. Various types of chalcone MAO inhibitors have been designed and synthesized. For example, a series of unsaturated chalcone derivatives have been synthesized that act as selective and reversible MAO-B inhibitors [32]. Selective inhibition of MAO-B was compared using fluorinated methoxylated chalcones and fluorinated chalcones containing morpholine and imidazole moieties [33, 34]. In addition, heterocyclic furanochalcone analogues [35], chlorinated thienyl chalcones [36], furanochalcones [37] and nitrocatechol chalcones [ 38] was investigated for its MAO-B inhibitory effect.

Recently, 4'-methoxy-4-dimethylaminochalcone [i.e., (2E)-3-[4-(dimethylamino)phenyl]-1-(4-methoxyphenyl) prop-2-en-1 -one] has been reported to strongly inhibit MAO-B (IC ₅₀ = 0.29 μM) [39], 4′-imidazole-4-dimethylaminochalcone [i.e., (2E)-3-[4-( Dimethylamino)phenyl]-1-[4-(1H-imidazol-1-yl)phenyl]prop-2-en-1-one] has been reported to effectively and nonselectively inhibit MAO-B ( IC ₅₀ = 6.2 μM) [40]. During this experiment, four 4-dimethylaminochalcone derivatives, namely 3'-trifluoromethyl-, 4'-bromo- and 4'-chloro-4-dimethylaminochalcone derivatives, were tested at concentrations of 0.150 to 0.539 μM. A study that selectively inhibits MAO-B with an IC ₅₀ value has been reported [41]. However, inhibition of MAO-A and MAO-B by 4-dimethylaminochalcone (Compound 2) and 2,4'-dichloro-4-dimethylaminochalcone (Compound 1) has not been previously reported. In this study, compound 2 (IC ₅₀ = 0.029 μM) inhibited MAO-B more potently than razabemide (0.046 μM; the reference reversible MAO-B inhibitor), and compound 1 (0.18 μM) inhibited MAO more than toloxatone. -A more potently (0.93 μM; the reference reversible MAO-A inhibitor). Compound 10 selectively inhibited MAO-B as previously reported (SI > 28.8), and compound 11 inhibited MAO-B more than MAO-A (SI > 3.03), contradicting previous results [ 23].

The chalcone in this experiment selectively inhibited MAO-B. That is, compound 2 inhibited MAO-B more strongly than compounds 3 and 4, which is a 4'-methoxy derivative of 4-dimethylaminochalcone, a more potent MAO-B inhibitor than 4'-chloro or 4'-nitro. It is reminiscent of what was found about [39]. Compound 2 was also found to be a reversible and competitive inhibitor of MAO-B common among chalcones [27, 37, 39, 41]. However, some natural chalcones such as isoliquiritigenin [28] and synthetic chalcones [42] have been reported to be selective for MAO-A. In this experiment, compound 12 (natural chalcone; 2,2'-dihydroxy-4',6'-dimethoxychalcone) was selective for MAO-A, but its potency was the most potent natural flavonoid against MAO-A. It was lower than that of one of the inhibitors, rhamnocitrin (IC ₅₀ = 0.051 μM) [9]. Of the endogenous β-carbolines, 7-methoxy harman (i.e., harmine) was more potent than Harman in MAO-A inhibitory activity [43].

Selectivity for either MAO-A or MAO-B is a very difficult problem, since the two isoforms have >70% sequence identity and function, calculated between Tyr 197 of MAO-A and the corresponding Tyr188 of MAO-B. This is because the calculated pKa value (up to 1.23 units) is large, but using the same mechanism with no substantial difference, suggesting that the electrostatic potential patterns of the two active sites are very similar [44]. The substrate and inhibitor specificity of an isoform is primarily influenced by the narrow part ("gate") defined by Ile199 and Tyr326 of the MAO-B cavity, but in general MAO-A It can accommodate larger compounds [45].

Dual inhibition of MAO-B and AChE has been investigated in the context of AD. In this study, five synthetic chalcones (compounds 1 to 5) effectively inhibited AChE with IC ₅₀ values ranging from 1.25 to 6.07 μM, and these 5 compounds exhibited better AChE than fluorinated chalcones with IC ₅₀ values >24.5 μM. effectively suppressed [34]. We propose that these five chalcones be considered candidates for dual function inhibitors to MAO-B and AChE.

A hydrophobic active site in MAOs has been proposed to favor unprotonated neutral substrates [47]. The IC ₅₀ values of compound 2 for MAO-A and MAO-B were lower than those of compound 4, and based on the property that the tertiary amine of compound 2 can only be protonated at low pH, the amino group might be in a neutral state. will be.

Regarding the docking assay, other results have observed a hydrogen bond interaction between the carbonyl oxygen of the chalcone and Cys172 of MAO-B [41]. Various R groups on C-4, including methyl, methoxy or dimethylamine, have been reported to increase the selective inhibition of MAO-B [32, 39]. Docking simulations of compound 6 with AChE predicted a hydrogen bonding interaction at Phe288, but the binding energy of this interaction was less than that of the other 11 compounds. Since the AutoDock Vina docking score was calculated considering hydrogen bonding, electrostatic bonding, van der Waals forces, and dissolution effects, it is thought that compound 6 must have interacted by hydrogen bonding and other contributions are low.

4. Conclusion

Among the 12 chalcone derivatives tested, 4-dimethylaminochalcone (Compound 2) inhibited human MAO-B most strongly with an IC ₅₀ value of 0.029, followed by 4'-chloro-4-dimethylaminochalcone (Compound 5). and 2,4′-dichloro-4-dimethylaminochalcone (Compound 1) with IC ₅₀ values of 0.061 and 0.075 μM, respectively. However, 4-carboxymethylchalcone (Compound 6) did not effectively inhibit MAO-B (IC ₅₀ = 10.6 μM). Compound 2 showed high selectivity for MAO-B (SI = 113.1), and compound 12 (2,2'-dihydroxy-4',6'-dimethoxychalcone) showed high selectivity for MAO-A (IC50 = 0.39 μM) was strongly and selectively inhibited. Interestingly, compound 2 reversibly and competitively inhibited MAO-B (Ki value = 0.0066 μM) and effectively inhibited AChE (IC50 = 6.07 μM). The binding affinity of the synthetic chalcone compounds 1 to 5 to MAO-B was higher than that to MAO-A or AChE, which formed a hydrogen bond with MAO-B at the Cys172 residue but with MAO-A or AChE. implying that no bond is formed. Our findings imply that Compound 2 and the like can be considered as MAO-B/AChE dual targeting inhibitors with potential for treatment of degenerative cranial nerve diseases such as Alzheimer's disease.

Claims (6)

Contains a compound selected from the group consisting of 2,4'-dichloro-4-dimethylaminochalcone, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a stereoisomer thereof as an active ingredient A pharmaceutical composition for the prevention or treatment of degenerative cranial nerve diseases.
According to claim 1, wherein the compound is to inhibit MAO-B (monoamine oxidase-B) and AChE (acetylcholinesterase), a pharmaceutical composition for the prevention or treatment of degenerative brain disease.
The method of claim 1, wherein the degenerative cranial nerve disease is at least one disease selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, learning disabilities, and memory impairment. Phosphorus, a pharmaceutical composition for the prevention or treatment of degenerative cranial nerve diseases.
Contains a compound selected from the group consisting of 2,4'-dichloro-4-dimethylaminochalcone, a pharmaceutically acceptable salt thereof, a hydrate thereof, and a stereoisomer thereof as an active ingredient A food composition for the prevention or improvement of degenerative cranial nerve diseases.
According to claim 4, wherein the compound is to inhibit MAO-B (monoamine oxidase-B) and AChE (acetylcholinesterase), a food composition for preventing or improving degenerative cranial nerve disease.
The method of claim 4, wherein the degenerative cranial nerve disease is at least one disease selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, learning disabilities, and memory impairment. Phosphorus, a food composition for preventing or improving degenerative cranial nerve diseases.

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