A system for the synthesis of novel chalcone analogues as major SARS-CoV-2 protease inhibitors

Abstract

A system for the synthesis of novel chalcone analogues as major protease inhibitors of SARS-CoV-2, consisting of: a reaction vessel configured to receive aldehyde and ketone precursors in ethanol solvent; a stirring mechanism that is operationally connected to the reaction vessel and configured to maintain continuous mixing of the reaction mixture at room temperature; a temperature control unit configured to maintain a specific temperature state during synthesis; a solvent supply mechanism configured to add sodium hydroxide solution dropwise to the reaction mixture; a filter arrangement for separating precipitates from the reaction mixture; a drying unit configured to remove residual solvents from filtered precipitate; and a recrystallization module configured to purify the obtained product using an ethanol solvent.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for synthesizing novel chalcone analogues as SARS-CoV-2 major protease inhibitors. More specifically, the present invention relates to a system for synthesizing novel chalcone analogues as SARS-CoV-2 major protease inhibitors and for characterizing the prepared chalcone analogues, as well as for conducting in silico studies of the prepared product, including docking, dynamics, and ADME profiling, to understand the physicochemical properties of the prepared compounds and evaluate their inhibitory potency against the SARS-CoV-2 major protease. BACKGROUND OF THE INVENTION

[0002] The emergence of SARS-CoV-2 and its rapid mutations have led to global health crises, necessitating the continued discovery of effective antiviral agents. Existing treatments, including remdesivir and hydroxychloroquine, have shown inconsistent efficacy, highlighting the need for novel therapeutics. Chalcone derivatives, known for their broad-spectrum biological activity, have demonstrated promising antiviral properties, including the inhibition of SARS-CoV-2-related infections. Given the increasing resistance of the virus to conventional drugs, the present invention focuses on the synthesis of chalcone-based compounds as potential inhibitors of the main protease of SARS-CoV-2.

[0003] In view of the above discussion, the present invention aims to provide a system for the synthesis of novel chalcone analogues as SARS-CoV-2 major protease inhibitors. Furthermore, the present invention aims to identify potent drug candidates with improved efficacy and minimal side effects through structural confirmation using 1 Dog 13 C-NMR as well as in silico molecular docking, dynamics and ADME studies. SUMMARY OF THE INVENTION

[0004] The present disclosure relates to a system for the synthesis of novel chalcone analogues as SARS-CoV-2 major protease inhibitors. The invention represents a comprehensive system for the synthesis of novel chalcone analogues as potential SARS-CoV-2 major protease inhibitors. The system integrates several modules, including reaction vessels, filtration devices, analytical components, and purification units, into a coherent platform that enables the controlled synthesis, isolation, and characterization of chalcone derivatives with potential antiviral properties.

[0005] This relates to providing a system for the synthesis of novel chalcone analogues as SARS-CoV-2 major protease inhibitors.The system comprises: a reaction vessel configured to accommodate aldehyde and ketone precursors in ethanol solvent; a stirring mechanism operatively connected to the reaction vessel and configured to maintain continuous mixing of the reaction mixture at room temperature; a temperature control unit configured to maintain a specific temperature condition during synthesis; a solvent supply mechanism configured to add sodium hydroxide solution dropwise to the reaction mixture; a filter assembly configured to separate precipitate from reaction mixture; a drying unit configured to remove residual solvent from filtered precipitate; and a recrystallization module configured to purify the resulting product with ethanol solvent.

[0006] An aim of the present disclosure is to provide a system for synthesizing novel chalcone analogues as major protease inhibitors of SARS-CoV-2.

[0007] Another aim of the present disclosure is to provide an integrated system for the efficient synthesis and purification of novel chalcone analogues with potential anti-SARS-CoV-2 activity.

[0008] Another aim of the present disclosure is to characterize and evaluate the efficacy of the prepared chalcone analogues.

[0009] A further objective of the present disclosure is to enable systematic quality control and characterization of synthesized compounds through integrated analytical functions.

[0010] 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

[0011] 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 novel chalcone analogues as SARS-CoV-2 major protease inhibitors according to an embodiment of the present disclosure; and Fig. Figure 2 illustrates a diagram and a table showing a scheme for preparing chalone analogues as major protease inhibitors of SARS-CoV-2 according to an embodiment of the present disclosure.

[0012] 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:

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0019] Fig. 1 shows a block diagram of a system (100) for synthesizing novel chalcone analogues as major protease inhibitors of SARS-CoV-2 according to an embodiment of the present disclosure.

[0020] With reference to Fig. 1, the system (100) comprises a reaction vessel (102) configured to contain aldehyde and ketone precursors in ethanol solvent; a stirring mechanism (104) operatively connected to the reaction vessel (102) and configured to maintain continuous mixing of the reaction mixture at room temperature; a temperature control unit (106) configured to maintain a specific temperature condition during synthesis; a solvent supply mechanism (108) configured to add sodium hydroxide solution dropwise to the reaction mixture; a filter assembly (110) configured to separate precipitate from the reaction mixture; a drying unit (112) configured to remove residual solvent from filtered precipitate;and a recrystallization module (114) configured to purify the resulting product using ethanol solvent;

[0021] In one embodiment, the reaction vessel (102) comprises: a round-bottomed flask (102a); a plurality of inlet ports (102b) for reagent addition; and a temperature monitoring sensor (102c).

[0022] In one embodiment, the filtration assembly (110) comprises: a Buchner funnel, a vacuum filtration mechanism, and a collection vessel configured to receive the filtered precipitate.

[0023] In one embodiment, the drying unit (112) comprises: a rotary evaporator, a vacuum system, and a temperature control mechanism configured to regulate the drying conditions.

[0024] In one embodiment, the system (100) further comprises an analytical module (116) configured to: determine melting points of the synthesized chalcone derivatives; measure NMR spectra; analyze chemical shifts and coupling constants. The analytical module (116) comprises: a Mel-Temp device (116a) for melting point determination; and a Bruker 400 spectrometer (116b) configured to operate at 400 MHz for 1- NMR and at 100 MHz for carbon 13 -NMR works.

[0025] In one embodiment, the system (100) further comprises a purification module (118) comprising: a chromatography column comprising 60-120 mesh silica gel; and a fraction collection system, wherein the purification module is configured to perform purification of the resulting product by column chromatography.

[0026] In one embodiment, the recrystallization module (114) comprises: a temperature-controlled crystallization vessel; a solvent delivery system configured to add minimal amounts of ethanol; and a crystal collection mechanism.

[0027] In one embodiment, the system (100) further comprises a quality control module (120) configured to verify product purity, analyze spectral data, and generate characterization reports.

[0028] In one embodiment, the system (100) is configured to maintain room temperature conditions during synthesis, monitor the reaction progress by precipitate formation, and control the dropwise addition rate of the sodium hydroxide solution.

[0029] The present invention relates to a system for the synthesis of chalcone analogues as major protease inhibitors of SARS-CoV-2. The system operates in sequential phases, with aldehyde and ketone precursors first combined in ethanol in a temperature-controlled reaction vessel. A controlled addition of sodium hydroxide solution initiates the synthesis reaction. The resulting precipitate is isolated by vacuum filtration using a Buchner funnel, followed by rotary evaporation for drying. The product is recrystallized for purification, and the final compound is characterized using integrated analytical modules such as NMR spectroscopy and melting point determination.

[0030] Fig. Figure 2 illustrates a diagram and a table showing a scheme for preparing chalcone analogues as major protease inhibitors of SARS-CoV-2 according to an embodiment of the present disclosure.

[0031] The system for the synthesis and purification of chalcone derivatives comprises a reaction vessel configured to accommodate aldehyde and ketone precursors dissolved in ethanol (EtOH) as a solvent. A stirring mechanism is operatively connected to the reaction vessel to maintain continuous mixing of the reaction mixture at room temperature. The system includes a temperature control unit to regulate and maintain the required temperature conditions throughout the synthesis process.

[0032] The system incorporates a solvent delivery mechanism that allows the dropwise addition of a freshly prepared sodium hydroxide (NaOH) solution in equivalent moles to the reaction mixture. During the reaction, a precipitate forms within 2-3 hours of stirring. To facilitate the precipitation process, an ice-cold Millipore water dispensing unit is configured to introduce water into the reaction vessel.

[0033] After precipitation, a filtration device equipped with a Buchner funnel is configured to separate the solid precipitate from the reaction mixture. The resulting solid product is then transferred to a drying unit, which uses a rotary evaporator to effectively remove the residual solvent, producing a purified, dry compound.

[0034] For further purification of the resulting product, a recrystallization module is integrated into the system. This module uses a minimal amount of ethanol solvent for recrystallization, thus ensuring a highly pure final product.

[0035] Analytical instruments are integrated into the system for quality control and characterization. The melting point of the synthesized chalcone derivatives is determined using a Mel-Temp device and recorded in degrees Celsius (uncorrected). The compounds are purified by column chromatography using 60-120 mesh silica gel. The structure of the synthesized chalcones is confirmed using nuclear magnetic resonance spectroscopy (NMR). A Bruker 400 spectrometer is used, operating at 400 MHz for 1 H-NMR and 100 MHz for 13C-NMR and uses the solvents CDCl3 and DMSO-d6. The system records chemical shifts (δ) in parts per million (ppm) and coupling constants (J) in Hertz (Hz), where 1 H-NMR signals can be categorized as singlet (s), doublet (d), triplet (t) or multiplet (m).

[0036] The system for molecular docking, molecular dynamics simulation, and ADMET analysis of chalcone derivatives is designed to facilitate the comprehensive evaluation of potential SARS-CoV-2 major protease inhibitors. The system includes an advanced computational docking module configured to analyze the binding affinity of the synthesized chalcone analogues (3a-c) in the active site of the SARS-CoV-2 major protease (PDB ID: 5N5O). The docking module integrates AutoDock Vina 1.1.2 within the PyRx software to ensure precise molecular interactions. The crystal structure of the SARS-CoV-2 major protease complexed with α-ketoamide (PDB ID: 5N5O, resolution: 2.00 Å, X-ray diffraction method) is retrieved from the Protein Data Bank. The docking module is also equipped with a molecular modeling interface that uses ChemDraw Professional 15.0 for ligand structure design and energy minimization.

[0037] A ligand binding analysis mechanism is integrated to identify the primary binding pocket using the Chimera program. This system identifies the key amino acid residues involved in the binding interaction, including Thr26, His41, Met49, Phe140, Leu141, Asn142, Gly143, Ser144, Cys145, His163, His164, Met165, Glu166, His172, Asp187, Gln189, and Thr190. The molecular docking system is configured with a search grid set to center coordinates of x: -23.158, y: 1.198, and z: 2.013 with size dimensions of x: 40, y: 40, and z: 40, maintaining a spacing of 0.375 Å. The completeness parameter is set to 8 to optimize docking calculations. Visualization of docking results is facilitated by the Maestro program, which allows for a detailed assessment of binding affinities.

[0038] The system also integrates a molecular dynamics (MD) simulation unit configured to evaluate the stability and dynamic behavior of the best-performing compound (SB4) in complex with the SARS-CoV-2 major protease (PDB ID: 5N5O). The MD simulation module uses the CHARMM36 force field and the GROMACS 2024 software package, enabling a 100 ns all-atom simulation. The system retrieves molecular topology files via the SwissParam online platform and includes an explicit water model to mimic physiological conditions. A charge neutralization unit is implemented that detects sodium (Na + ) and chloride ions (Cl -) to maintain system equilibrium. The MD simulation system consists of a structure minimization module operating under the CHARMM36 force field, followed by an equilibration unit that maintains a temperature of 310 K in the NPT ensemble for 100 seconds. The Lincs technique is employed to confine hydrogen atoms, while van der Waals interactions are calculated using a switching method within a cutoff of 12–14 Å.

[0039] A long-range electrostatics calculation module is integrated, which uses the Particle Mesh-Ewald (PME) method to ensure accurate force calculations. The system also includes a barostat control unit to adjust the system size at a target pressure of 1 bar while maintaining a constant temperature of 310 K. The simulation process is executed with 50,000,000 steps and an integration time step of 2 fs, resulting in a total runtime of 100 ns. Upon completion, the trajectory data are analyzed via an integrated visualization and analysis module using XMGRACE (version 5.1.19). This module evaluates key molecular properties, such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonding (H-bonding), radius of gyration (Rg), and solvent accessible surface area (SASA), to determine molecular stability and interaction efficiency.

[0040] The system also features an ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis module designed to evaluate the pharmacokinetic properties and toxicity risks of synthesized compounds. The ADMET module is configured to process molecular descriptors and predict bioavailability, metabolic stability, and potential toxic effects. This ensures the selection of promising drug candidates with optimized efficacy and minimal side effects.

[0041] The synthesized chalcone analogues 3(ac) were successfully obtained using aldehyde and ketone precursors in ethanol with NaOH as a catalyst. The reaction yielded solid products, which were purified by recrystallization. Their structures were confirmed by 1H and 13C NMR analysis.

[0042] Molecular docking analysis revealed that redocking of the co-crystallized ligand maintained key interactions with HIS 41, GLN 189, GLU 166, MET 49, and CYS 145, confirming the docking protocol. All synthesized chalcone analogues exhibited binding affinities between -5.4 and -6.2 kcal / mol, indicating lower affinity than the co-crystallized ligand and remdesivir, but comparable or slightly better values ​​than hydroxychloroquine. Among them, compound 3a had the highest docking score (-6.2 kcal / mol), followed by 3c (-5.8 kcal / mol), indicating their potential as inhibitors of the major protease of SARS-CoV-2. The docking interactions of compound 3a and the co-crystallized ligand with the target protein were analyzed and visualized.

[0043] Molecular dynamics simulations were performed to evaluate the stability and dynamic behavior of the protein-ligand complexes. RMSD analysis showed that the co-crystallized ligand and the 3a complex exhibited initial fluctuations (1–3 Å), but both stabilized over time within an acceptable range of less than 3 Å, confirming structural stability. RMSF analysis revealed significant fluctuations at the N-terminal and C-terminal regions, with the 3a complex fluctuating between 1 and 2 Å, except for a peak at approximately 4 Å in residues 2700–3000. Analysis of the radius of gyration (Rg) showed that the apo protein structure had the lowest Rg value, while the co-crystallized complex and the 3a complex showed fluctuations and stabilized at approximately 24 Å and 23 Å, respectively, suggesting that 3a maintained a stable protein structure.These results were further confirmed by SASA analysis, with the apo structure showing the lowest SASA value (~150 nm. 2 ) and the SB4 complex the highest (~225 nm 2 ), indicating differences in protein compactness.

[0044] The ADMET analysis evaluated the druglikeness and pharmacokinetic properties of the synthesized compounds. All compounds showed high gastrointestinal absorption, indicating good oral bioavailability. Compounds 3a, 3b, and 3c were predicted to cross the blood-brain barrier, indicating potential CNS activity. They were all substrates of cytochrome P450 enzymes and were predicted to inhibit CYP1A2, CYP2C19, and CYP2C9, which are critical for drug metabolism. Lipinski's 5-rule analysis confirmed that none of the compounds violated more than one rule, suggesting favorable druglike properties, with the exception of SB4, which violated only one rule but remained within acceptable limits. Furthermore, none of the compounds exhibited PAINS (Pan-Assay Interference Compounds), indicating minimal off-target effects.Clearance predictions ranged from 5 to 12 ml / min / kg, indicating moderate elimination rates suitable for maintaining effective drug concentrations.

[0045] Overall, the synthesized chalcone derivatives demonstrated promising inhibitory potential against the major protease of SARS-CoV-2, with 3a showing the best performance. Their stability in molecular dynamics, favorable binding interactions, and acceptable pharmacokinetic properties underscore their potential as drug candidates for further investigation.

[0046] 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.

[0047] 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 -Block diagram of a system for the synthesis of novel chalcone analogues as SARS-CoV-2 major protease inhibitors. 102 reaction vessel 102a round-bottom flask 102b Multiple inlet openings 102c Temperature monitoring sensor 104 agitator 106 Temperature control device 108 Solvent supply mechanism 110 filter system 112 Drying unit 114 Recrystallization module 116 An Analytical Module 116a Mel-Temp Apparatus 116b Bruker 400 spectrometer 118 Cleaning module 120 Quality Control Module

Claims

[1] A system for the synthesis of novel chalcone analogues as major protease inhibitors of SARS-CoV-2, consisting of: a reaction vessel configured to contain aldehyde and ketone precursors in ethanol solvent; a stirring mechanism operatively connected to the reaction vessel and configured to maintain continuous mixing of the reaction mixture at room temperature; a temperature control unit configured to maintain a specific temperature condition during synthesis; a solvent supply mechanism configured to add sodium hydroxide solution dropwise to the reaction mixture; a filter arrangement for separating precipitates from the reaction mixture; a drying unit configured to remove residual solvent from filtered precipitate; and a recrystallization module configured to purify the resulting product using an ethanol solvent. [2] The system of claim 1, wherein the reaction vessel comprises: a round-bottomed flask, a plurality of inlet ports for reagent addition, and a temperature monitoring sensor. [3] The system of claim 1, wherein the filter assembly comprises: a Buchner funnel, a vacuum filter mechanism, and a collection vessel configured to receive the filtered precipitate. [4] The system of claim 1, wherein the drying unit comprises: a rotary evaporator, a vacuum system, and a temperature control mechanism configured to regulate the drying conditions. [5] The system of claim 1, further comprising an analysis module configured to determine the melting points of the synthesized chalcone derivatives, measure NMR spectra, and analyze chemical shifts and coupling constants. [6] The system of claim 5, wherein the analytical module comprises: a Mel-Temp instrument for melting point determination; and a Bruker 400 spectrometer capable of operating at 400 MHz for 1- NMR and 100 MHz for carbon -13- NMR is configured. [7] The system of claim 1, further comprising a purification module comprising: a chromatography column comprising silica gel of 60-120 mesh; and a fraction collection system, wherein the purification module is configured to perform the purification of the obtained product by column chromatography. [8] The system of claim 1, wherein the recrystallization module comprises: a temperature-controlled crystallization vessel; a solvent dispensing system configured to add minimal amounts of ethanol; and a crystal collection mechanism. [9] The system of claim 1, further comprising a quality control module configured to verify product purity, analyze spectral data, and generate characterization reports. [10] The system of claim 1, wherein the system is configured to: maintain room temperature conditions during synthesis, monitor the progress of the reaction by precipitate formation, and control the dropwise addition rate of the sodium hydroxide solution.

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