A system for the production of piperazine-based chalcones as monoamine oxidase B inhibitors

Abstract

A system (100) for synthesizing piperazine-based chalcones as monoamine oxidase B inhibitors, comprising: a reaction vessel unit (102) configured to maintain controlled mixing conditions for combining 4'-piperazinoacetophenone with aromatic benzaldehyde; a solvent dispensing unit (104) configured to dispense predetermined amounts of ethanol and potassium hydroxide solution; a stirring unit (106) configured to maintain continuous motion for 10-12 hours; a precipitation unit (108) comprising an ice-cold water bath for product collection; a cleaning unit (110) configured for washing and recrystallizing the precipitate using ethanol; and a product analysis unit (112) configured to perform thin-layer chromatography using an ethyl acetate-hexane solvent system.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for synthesizing piperazine-based chalcones as monoamine oxidase B inhibitors. More specifically, the present invention relates to a synthesis of piperazine derivatives containing 1,3-diphenylprop-2-en-1-one derivatives (PC1-PC11) and the evaluation of their inhibitory activities against monoamine oxidases (MAOs), cholinesterases (ChEs), and β-site amyloid precursor protein-cleaving enzyme 1 (BACE-1) with a view to developing new treatments for neurological diseases. BACKGROUND OF THE INVENTION

[0002] Neurodegenerative diseases (NDDs), particularly Alzheimer's disease (AD) and Parkinson's disease (PD), pose a significant health challenge for the aging population. These diseases are caused by multiple factors, including genomic, epigenomic, and environmental influences. Traditional single-target therapeutic approaches have proven inadequate to treat the complex pathophysiology of NDDs, which include oxidative stress, mitochondrial dysfunction, and irregular biogenic amine metabolism.

[0003] Current drug development strategies have focused on creating compounds that can simultaneously target multiple enzymes involved in NDD progression, particularly cholinesterase (ChE), monoamine oxidases (MAOs), and β-site amyloid precursor protein-cleaving enzyme 1 (BACE-1). Piperazine, a preferred structure in pharmaceutical chemistry, has emerged as a promising scaffold due to its flexibility and ability to form both hydrophobic and hydrogen bonds. Currently, numerous FDA-approved drugs in various therapeutic categories contain the piperazine structure.

[0004] Similarly, chalcones, characterized by their 1,3-diphenylprop-2-en-1-one structure, have demonstrated selective MAO-B inhibitory properties. While various chalcone analogues with different heterocyclic moieties have been investigated as MAO-B inhibitors, the potential of combining piperazine and chalcone pharmacophores for the treatment of NDD remains unexplored. This invention closes this gap and provides a system for the synthesis and evaluation of novel piperazine-derived chalcones as multi-target inhibitors for the treatment of neurodegenerative diseases. SUMMARY OF THE INVENTION

[0005] The present disclosure relates to a system for the synthesis of piperazine-based chalcones as monoamine oxidase-b inhibitors. The present invention provides a comprehensive system for the synthesis and characterization of novel piperazine-based chalcones by base-catalyzed Claisen-Schmidt condensation. The system comprises integrated units for the synthesis, purification, and multifaceted analysis of the compounds. The core synthesis unit efficiently combines 4'-piperazinoacetophenone with aromatic benzaldehydes under controlled conditions, followed by specialized units for product isolation and purification. The system also includes advanced analytical capabilities for enzyme inhibition studies, particularly with a focus on MAO and cholinesterase activities, as well as molecular modeling capabilities for understanding binding interactions.

[0006] The present disclosure is intended to provide a system for synthesizing piperazine-based chalcones as monoamine oxidase B inhibitors. The system comprises: a reaction vessel unit configured to maintain controlled mixing conditions for the combination of 4'-piperazinoacetophenone with aromatic benzaldehyde; a solvent dispensing unit configured to dispense predetermined amounts of ethanol and potassium hydroxide solution; a stirring unit configured to maintain continuous agitation for 10 to 12 hours; a precipitation unit including an ice-cold water bath for product collection; a purification unit configured to wash and recrystallize the precipitate with ethanol; and a product analysis unit configured to perform thin-layer chromatography using an ethyl acetate-hexane solvent system.

[0007] An object of the present disclosure is to provide a system for the synthesis of piperazine-based chalcones as monoamine oxidase B inhibitors.

[0008] Another object of the present disclosure is an efficient and integrated system for the synthesis of piperazine-derived chalcones by controlled base-catalyzed Claisen-Schmidt condensation reactions, including precise reagent delivery, reaction monitoring, and product purification capabilities.

[0009] A further objective of the present disclosure is to establish a comprehensive analytical platform within the system to evaluate the enzyme inhibitory activity of the synthesized compounds, in particular against MAO and cholinesterase, together with molecular modeling capabilities to understand drug-target interactions.

[0010] A further objective of the present disclosure is to provide a modular system design that enables both the synthesis and characterization of potential multi-target inhibitors for neurodegenerative diseases through integrated reaction, purification and analysis units.

[0011] To further clarify the advantages and features of the present disclosure, a more detailed description of the invention will be given with reference to specific embodiments thereof illustrated in the accompanying drawings. It should be noted that these drawings represent only typical embodiments of the invention and are therefore not to be considered limiting its scope. The invention will be described and explained in additional detail and in greater detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE CHARACTERS

[0012] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. 1 shows a block diagram of a system for synthesizing piperazine-based chalcones as monoamine oxidase b inhibitors according to an embodiment of the present disclosure; and Fig. Figure 2 shows a diagram of Scheme 1 illustrating the synthetic route used in this invention according to one embodiment of the present disclosure.

[0013] Furthermore, those skilled in the art will appreciate that elements in the drawings are shown for convenience and may not necessarily be drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Moreover, with respect to device construction, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art who would benefit from the description herein. DETAILED DESCRIPTION:

[0014] To facilitate an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and described in specific language. It is to be understood, however, that this is not intended to limit the scope of the invention, as such changes and further modifications to the illustrated system and such further applications of the principles of the invention as illustrated therein are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0015] It will be understood by one skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0016] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, occurrences of the phrase "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not all refer to the same embodiment.

[0017] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only includes those steps, but may also include other steps not expressly listed or inherent in such process or method. Likewise, one or more devices or subsystems or elements or structures or components preceded by "comprises...a" does not preclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures or other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0019] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0020] Fig. 1 shows a block diagram of a system (100) for synthesizing piperazine-based chalcones as monoamine oxidase B inhibitors according to an embodiment of the present disclosure.

[0021] With reference to Fig. 1, the system (100) comprises a reaction vessel unit (102) configured to maintain controlled mixing conditions for combining 4'-piperazinoacetophenone with aromatic benzaldehyde; a solvent dispensing unit (104) configured to dispense predetermined amounts of ethanol and potassium hydroxide solution; an agitation unit (106) configured to maintain continuous agitation for 10-12 hours; a precipitation unit (108) comprising an ice-cold water bath for collecting the product; a purification unit (110) configured to wash and recrystallize the precipitate using ethanol; and a product analysis unit (112) configured to perform thin-layer chromatography using an ethyl acetate-hexane solvent system.

[0022] In one embodiment, the system (100) further comprises an enzyme assay unit (114) configured to analyze the inhibitory activities of the enzymes monoamine oxidase (MAO) and cholinesterase.

[0023] In one embodiment, the system (100) further comprises a kinetic analysis unit (116) configured to IC 50 -values ​​and to conduct enzyme inhibition studies.

[0024] In one embodiment, the system (100) further comprises a reversibility analysis unit (118) configured to assess the reversibility of enzyme inhibition by dialysis processes.

[0025] In one embodiment, the system (100) further comprises a molecular modeling unit (120) configured to perform molecular docking simulations and binding energy calculations.

[0026] In one embodiment, the reaction vessel unit (102) comprises temperature control mechanisms and a mixing device for maintaining uniform reaction conditions.

[0027] In one embodiment, the enzyme testing unit (114) comprises spectrophotometric detection systems for measuring enzyme activities.

[0028] In one embodiment, the molecular modeling unit (120) comprises computer hardware configured to perform molecular mechanics calculations and binding free energy determinations.

[0029] Fig. Figure 2 shows a diagram of Scheme 1 illustrating the synthetic route used in this invention according to one embodiment of the present disclosure.

[0030] In relation to Fig. 2, the derivatives (PC1-PC11) were prepared using a base-catalyzed Claisen-Schmidt condensation reaction between various aromatic aldehydes and 4'-piperazinoacetophenone.

[0031] With reference to Fig. 2 The following table shows the eleven synthesized derivatives. Table 2: Eleven derivatives. CODE R PC1 H PC2 OH PC3 OCH3 PC4 CH3 PC5 N(CH3)2 PC6 C2H5 PC7 NO2 PC8 Cl PC9 Br PC10 F PC11 CF3

[0032] The synthesis of the derivatives began by dissolving 0.01 M 4'-piperazinoacetophenone in a mixture of ethanol (20 mL) and 40% KOH (8 mL). Subsequently, 0.01 M of an aromatic benzaldehyde was added, and the reaction mixture was stirred continuously for 10–12 hours (Scheme 1). The resulting mixture was then poured into ice-cold water to precipitate the products, which were then thoroughly washed with water. The formation of the desired products was confirmed by thin-layer chromatography (TLC) using a solvent system of ethyl acetate and hexane in a ratio of 1:9. Finally, the products were purified by recrystallization with ethanol.

[0033] Enzyme activity assays were performed using kynuramine (0.06 mM) and benzylamine (0.3 mM) as substrates to evaluate the activities of recombinant human MAO-A and MAO-B, respectively. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured using Electrophorus electricus type VI-S and equine serum type, respectively. The assays included 0.5 mM acetylthiocholine iodide (ATCI) or 0.05 mM butyrylthiocholine iodide (BTCI) as substrates, combined with 0.5 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Enzymes and inhibitors were pre-incubated for 15 minutes before measuring their inhibitory activities. A β-secretase (BACE-1) activity detection kit was used for BACE-1 activity. The substrate of the kit was 7-methoxycoumarin-4-acetyl-[Asn670, Leu671]-amyloid β / A4 protein fragment 667-676-(2,4-dinitrophenyl)Lys - Arg - argamide trifluoroacetate.The reaction was carried out for 2 hours at 37 °C and the fluorescence signals were measured using a Scinco FS-2 fluorescence spectrometer (Seoul, Korea) with excitation and emission wavelengths of 320 nm and 405 nm, respectively.

[0034] The inhibitory activities of 11 compounds were initially investigated at a concentration of 10 µM against MAO-A, MAO-B, AChE, BChE, and BACE-1. Compounds with residual activities below 50% were further investigated to determine their IC50 values ​​first for MAO-A and MAO-B, and then for AChE, BChE, and BACE-1. Kinetic studies were performed on PC10 and PC11, the most potent inhibitors of MAO-B, using five different substrate concentrations and three inhibitor concentrations.

[0035] The reversibility of MAO-B inhibition by PC10 and PC11 was assessed by dialysis after pre-incubation with 0.15 µM MAO-B for 30 minutes. For comparative analysis, MAO-B was also pre-incubated with lazabemide, a reversible reference MAO-B inhibitor, at a concentration of 0.20 µM, and pargyline, an irreversible reference MAO-B inhibitor, at a concentration of 0.30 µM. Reversibility was determined by analyzing the enzyme activity of dialyzed (AD) and non-dialyzed (AU) samples.

[0036] For ADME prediction, the ADME parameters including pharmacokinetic data and physicochemical properties such as lipophilicity and water solubility of PC10 and PC11 were predicted.

[0037] Molecular docking was performed using the 3D coordinates of the MAO-A, MAO-B, and AChE crystal structures retrieved from the Protein Data Bank under entries 2Z5X, 2V5Z, and 4EY7, respectively. The Protein Preparation Wizard tools were used to optimize and minimize the crystal structures. To account for chirality, ionization states, ring conformations, and tautomeric forms of the input structures, the LigPrep tool was used to treat PC4, PC10, and PC11. Docking simulations were performed using GLIDE, with the enclosing boxes defined by the centers of gravity of related ligands. The standard precision docking protocol was applied using the OPLS_2005 force field, followed by a detailed analysis of ligand binding affinities.Binding free energies (ΔG) between proteins and ligands were calculated using the Molecular Mechanics / Generalized Born Surface Area (MM-GBSA) method. ΔGbind values ​​were calculated using the formula ΔG . bind = ΔE MM + ΔG solv + ΔG SA derived, where ΔE MM represents the minimized energy of the ligand-protein complex, ΔG solv the solvation energy is taken into account and ΔG SA corresponds to the surface energy.

[0038] In this invention, the inhibitory activities of eleven compounds (PC1-PC11) against MAO-B, MAO-A, AChE, BChE, and BACE-1 were analyzed. The results showed that all compounds exhibited potent inhibition of MAO-B, with residual activities below 50% at a concentration of 10 µM. Among them, PC10 and PC11 showed the strongest inhibition with IC50 values ​​of 0.65 µM and 0.71 µM, respectively, while PC1 showed the weakest inhibitory effect with an IC50 of 7.62 µM. The analysis revealed that substitutions such as -F or -CF3 significantly enhanced MAO-B inhibition, while other groups such as -OCH3, -Cl, and -OH moderately increased the activity. In contrast, these compounds were much less effective against MAO-A, with residual activities of over 65% at the same concentration, and the lowest observed IC 50 The α-value was 27.9 µM for PC3. PC10 and PC11 showed high selectivity for MAO-B over MAO-A, which was reflected in their selectivity indices.

[0039] The compounds generally showed weak to moderate inhibition of AChE and BChE, with most achieving inhibition values ​​below 50% at 10 µM. However, PC4 was an exception, inhibiting AChE by 56.6% with an IC50 of 8.77 µM. PC10 and PC11 showed moderate inhibitory potency against both enzymes. Notably, certain compounds, including PC3, PC10, and PC11, potently inhibited BACE-1, with IC50 values ​​indicating moderate potency for PC10 and PC11. The presence of fluoro- and trifluoromethyl pharmacophores on their piperazine-substituted chalcone scaffolds appeared to contribute to their activity.

[0040] Kinetic studies showed that PC10 and PC11 competitively inhibited MAOI, with Ki values ​​of 0.63 ± 0.13 µM and 0.53 ± 0.068, respectively. Reversibility tests demonstrated that MAOI inhibition by these compounds was reversible, with recovery rates comparable to those of the reversible reference inhibitor lazabemide, in contrast to the irreversible inhibitor pargyline. This finding confirmed the reversible and selective inhibitory nature of PC10 and PC11.

[0041] The results of the reversibility studies showed that MAO-B inhibition by PC10 and PC11 was restored to reversible reference values ​​and demonstrated that PC10 and PC11 reversibly inhibit MAO-B.

[0042] In silico ADME predictions showed that both PC10 and PC11 conformed to Lipinski's rule of five, supporting their druglike nature and suitability for oral administration. The boiled egg and bioavailability radar charts indicated their ability to cross the blood-brain barrier and be passively absorbed gastrointestinally. While PC10 differed slightly in terms of saturation, both compounds were deemed suitable for oral administration based on their physicochemical properties.

[0043] Molecular docking studies provided insights into the interactions of PC10 and PC11 with enzyme binding sites. Both compounds exhibited stronger binding affinities for MAO-B compared to MAO-A, with docking scores and MM-GBSA values ​​supporting these observations. Similar binding positions within the MAO-B binding pocket were observed for these compounds, involving π-π interactions with key residues. For AChE, PC4 exhibited the lowest binding free energy and unique interactions, including hydrogen bonds, that differentiated its activity from that of PC10 and PC11. These findings highlight the potential of PC10 and PC11 as promising lead compounds for selective MAO-B inhibition and their moderate inhibitory effects on other enzymes.

[0044] The results showed that all eleven compounds inhibited MAO-B more potently than MAO-A, and compounds PC4, PC10, and PC11 potentially inhibited MAO-B with IC50 values ​​of 2.72, 0.65, and 0.71 µM, respectively, and moderately inhibited AChE with IC50 values ​​of 8.77, 28.0, and 26.3 µM, respectively. Furthermore, these experimental results were also supported by molecular docking studies. Furthermore, PC4, PC10, and PC11 moderately inhibited BACE-1 with IC50 values ​​of 15.5, 14.9, and 15.3 µM, respectively, further supporting their potential use for the development of new drugs against various neurological diseases.

[0045] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of the processes described herein may be changed and are not limited to the manner described herein. Furthermore, the actions of any flowchart need not be implemented in the order shown; nor do all actions necessarily need to be performed. Also, those actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and use of materials, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0046] Advantages, other benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, benefits, solutions to problems, and any components that may cause an advantage or solution to occur or become more apparent are not to be construed as a critical, required, or essential feature or component of any or all of the claims. REFERENCES 100 A system for the synthesis of piperazine-based chalcones as monoamine oxidase B inhibitors. 102 Reaction vessel unit 104 Solvent dosing unit 106 agitator 108 precipitation units 110 cleaning system 112 Product Analysis Unit 114 Enzyme Test Unit 116 Kinetic Analysis Unit 118 Reversibility Analysis Unit 120 Molecular Modeling Unit

Claims

[1] A system (100) for synthesizing piperazine-based chalcones as monoamine oxidase B inhibitors, comprising: a reaction vessel unit (102) configured to maintain controlled mixing conditions for combining 4'-piperazinoacetophenone with aromatic benzaldehyde; a solvent dispensing unit (104) configured to dispense predetermined amounts of ethanol and potassium hydroxide solution; a stirring unit (106) configured to maintain continuous agitation for 10-12 hours; a precipitation unit (108) comprising an ice-cold water bath for product collection; a purification unit (110) configured to wash and recrystallize the precipitate using ethanol; and a product analysis unit (112) configured to perform thin layer chromatography using an ethyl acetate-hexane solvent system. [2] The system of claim 1, further comprising an enzyme assay unit (114) configured to analyze the inhibitory activities of the enzymes monoamine oxidase (MAO) and cholinesterase. [3] The system of claim 1, further comprising a kinetic analysis unit (116) configured to IC 50 -values ​​and to conduct enzyme inhibition studies. [4] The system of claim 1, further comprising a reversibility analysis unit (118) configured to assess the reversibility of enzyme inhibition by dialysis processes. [5] The system of claim 1, further comprising a molecular modeling unit (120) configured to perform molecular docking simulations and binding energy calculations. [6] The system of claim 1, wherein the reaction vessel unit (102) includes temperature control mechanisms and a mixing device for maintaining uniform reaction conditions. [7] The system of claim 2, wherein the enzyme testing unit (114) comprises spectrophotometric detection systems for measuring enzyme activities. [8] The system of claim 5, wherein the molecular modeling unit (120) comprises computer hardware configured to perform molecular mechanics calculations and binding free energy determinations.

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