A system for the synthesis of methylsulfonylacrylimidamide analogues

Abstract

System (100) for the synthesis of new methylsulfonylacrylimidamide analogues, comprising: a) a reaction vessel unit (102) configured to maintain a controlled reaction environment; b) a reagent supply unit (104) configured to introduce the following sequentially: - Propargyl bromide - secondary amine, - Triethylamine, - Water, - Mesylazide and - Copper iodide catalyst; c) a reaction monitoring unit (106) configured to: - to detect the development of nitrogen gas, and - to monitor the completion of the reaction using thin-layer chromatography; d) a temperature control unit (108) configured to maintain room temperature conditions during the reaction; and e) a stirring unit (110) in conjunction with the reaction vessel unit (102) configured to ensure continuous stirring of the reaction mixture.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for synthesizing methylsulfonylacrylimidamide analogues. More specifically, the present invention relates to a system for synthesizing a series of novel methylsulfonylacrylimidamide analogues (4 a-4 h) and screening them for their α-glucosidase-inhibiting activity. BACKGROUND OF THE INVENTION

[0002] Type 2 diabetes mellitus (T2DM) affects approximately 537 million adults worldwide, and this number is expected to rise to 643 million by 2045. Current α-glucosidase inhibitors such as miglitol, acarbose, and voglibose are effective in treating T2DM, but cause significant side effects such as bloating and abdominal discomfort due to poor absorption rates.

[0003] α-Glucosidase, located in the brush border of the small intestine, plays a crucial role in carbohydrate digestion by breaking down polysaccharides into glucose. Its inhibition contributes to the maintenance of normal blood glucose levels, making it an important therapeutic target for the treatment of T2DM.

[0004] The synthesis of methylsulfonylacrylimidamide derivatives using click chemistry represents a novel approach for the development of more potent α-glucosidase inhibitors. Acrylamidines, which serve as key precursors in this synthesis, have demonstrated promising pharmacological activities, including insulin-releasing properties. The present invention aims to overcome the limitations of existing treatments by providing a system configured for the development of more specific, targeted inhibitors with minimal side effects for the treatment of T2DM. SUMMARY OF THE INVENTION

[0005] The present disclosure relates to a system for the synthesis of methylsulfonylacrylimidamide analogues. The invention relates to a system for the synthesis of novel methylsulfonylacrylimidamide derivatives via a click chemistry approach for the inhibition of α-glucosidase. The system is configured to enable a copper-catalyzed reaction between propargyl bromide, secondary amines, and mesyl azide to produce methylsulfonylacrylimidamide analogues exhibiting significant α-glucosidase inhibitory activity with IC50 values ​​exceeding those of the standard drug acarbose.

[0006] The present disclosure aims to provide a system for synthesizing methylsulfonylacrylimidamide analogues. The system comprises: a reaction vessel unit configured to maintain a controlled reaction environment; a reagent feed unit configured to sequentially introduce propargyl bromide, secondary amine, triethylamine, water, mesyl azide, and copper iodide catalyst; a reaction monitoring unit configured to detect the evolution of nitrogen gas and monitor reaction completion via thin-layer chromatography; a temperature control unit configured to maintain room temperature conditions during the reaction; and a stirring unit connected to the reaction vessel unit and configured to continuously stir the reaction mixture.

[0007] An object of the present disclosure is to provide a system for synthesizing methylsulfonylacrylimidamide analogues.

[0008] Another aim of the present disclosure is the synthesis and characterization of novel methylsulfonylacrylimidamide derivatives using click chemistry for the potential treatment of type 2 diabetes mellitus.

[0009] A further objective of the present disclosure is to evaluate the α-glucosidase inhibitory activity of the synthesized compounds and to assess their cytotoxicity against human cell lines.

[0010] Another objective of the present disclosure is to investigate the druglikeness of the compounds through ADME studies and molecular docking analyses.

[0011] To further clarify the advantages and features of the present disclosure, a more particular description of the invention will be provided by reference to specific embodiments illustrated in the accompanying drawings. These drawings are understood to represent only typical embodiments of the invention and are therefore not to be considered limiting the scope of the invention. The invention will be described and explained with additional specificity and detail with the accompanying drawings. BRIEF DESCRIPTION OF THE ILLUSTRATIONS

[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 the drawings, wherein: Fig. 1 shows a block diagram of a system for synthesizing methylsulfonylacrylimidamide analogues according to an embodiment of the present disclosure; Fig. Figure 2 is a diagram illustrating the scheme for synthesizing methylsulfonylacrylimidamide according to an embodiment of the present disclosure; and Fig. Figure 3 shows a diagram illustrating the plausible mechanism for the formation of methylsulfonylacrylamidine in accordance with one embodiment of the present disclosure.

[0013] Those skilled in the art will understand that the elements in the drawings are shown for convenience and are not necessarily drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, one or more components of the apparatus may be represented in the drawings by conventional symbols, and the drawings may show only the 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 familiar with the present description. DETAILED DESCRIPTION:

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

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

[0016] When this specification refers to "one aspect," "another aspect," or the like, it means 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, the terms "in one embodiment," "in another embodiment," and similar expressions throughout this specification may or may not all refer to the same embodiment.

[0017] The terms "comprises," "including," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only comprises those steps, but may also include other steps not expressly listed or included in such process or method. Likewise, one or more devices or subsystems or elements or structures or components introduced with "comprises...a" do 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 otherwise defined, 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 are not intended to be 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 methylsulfonylacrylimidamide analogues according to an embodiment of the present disclosure.

[0021] Referring to Fig. 1, the system (100) comprises a reaction vessel unit (102) configured to maintain a controlled reaction environment; a reagent feed unit (104) configured to sequentially introduce propargyl bromide, secondary amine, triethylamine, water, mesyl azide, and copper iodide catalyst; a reaction monitoring unit (106) configured to detect the evolution of nitrogen gas and monitor the completion of the reaction via thin-layer chromatography; a temperature control unit (108) configured to maintain room temperature conditions during the reaction; and a stirring unit (110) in communication with the reaction vessel unit (102) configured to enable continuous stirring of the reaction mixture.

[0022] In one embodiment, the system (100) further comprises a characterization unit (112) configured to perform proton nuclear magnetic resonance spectroscopy at 400 MHz, carbon-13 nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, and chemical shift and coupling constant measurements.

[0023] In one embodiment, the system (100) further comprises an analysis unit (114) configured to measure α-glucosidase inhibitory activity, determine IC50 values, and perform cytotoxicity assessments against human cell lines.

[0024] In one embodiment, the reagent delivery unit (104) is configured to maintain precise stoichiometric ratios of 0.5 millimoles of propargyl bromide, 0.5 millimoles of secondary amine, 0.6 millimoles of triethylamine, 0.6 millimoles of mesyl azide, and 0.05 millimoles of copper iodide.

[0025] In one embodiment, the system (100) further comprises a computational analysis unit (116) configured to perform molecular docking studies, make ADME predictions, and verify compliance with Lipinski's rule of five.

[0026] In one embodiment, the system (100) further comprises a quality control unit (118) configured to monitor reaction progress using thin layer chromatography, verify product purity, and validate spectroscopic data.

[0027] In one embodiment, the characterization unit (112) is configured to use residual solvent signals as internal references for nuclear magnetic resonance measurements.

[0028] In one embodiment, the analysis unit (114) is configured to compare the inhibitory activities with the standard acarbose.

[0029] The present invention relates to a system for the synthesis of methylsulfonylacrylimidamide analogues (4a-4h) by a room-temperature reaction involving propargyl bromide, secondary amines, and mesyl azide in the presence of a copper iodide catalyst. The synthesized compounds exhibit potent α-glucosidase inhibition, with compounds 4f and 4h exhibiting particularly high activity. The compounds exhibit low cytotoxicity and favorable ADME properties, making them promising candidates for diabetes treatment.

[0030] Fig. 2 shows a diagram illustrating the scheme for synthesizing methylsulfonylacrylimidamide according to an embodiment of the present disclosure.

[0031] As in Fig. As shown in Figure 2, compounds 4a-4h were prepared using a bioisosteric approach through a Cu-catalyzed multicomponent reaction of mesyl azide, alkyne, and secondary amine in water.

[0032] For the synthesis of the aforementioned compound (4a-4h), propargyl bromide (0.5 mmol) was added to a round-bottom flask, followed by the sequential addition of a secondary amine (0.5 mmol), triethylamine (0.6 mmol), and 5 mL of water. Subsequently, mesyl azide (0.6 mmol) was added to the same reaction mixture, followed by the addition of CuI (0.05 mmol), which resulted in the evolution of N2 gas. The reaction mixture was stirred at room temperature until complete consumption of the reactants, which was confirmed by TLC analysis.

[0033] Thin layer chromatography (TLC) was performed on 0.20 mm silica gel plates. Proton ( 1 H) and carbon ( 13C) NMR spectra were recorded with a 400 MHz Bruker spectrometer, using residual solvent signals as internal references. Chemical shifts (δ) are given in ppm (parts per million), and coupling constants (J) are given in Hz (hertz). The following notations are used: s (singlet), m (multiplet), brs (broad singlet), d (doublet), t (triplet), q (quartet). Fourier transform infrared (FT-IR) spectra were measured with a Bruker α-spectrophotometer and are expressed in wavenumbers (cm- 1 ) is indicated.

[0034] The good synthetic accessibility of methylsulfonylacrylimidamide is due to the commercial availability of structurally diverse mesyl azides, alkynes, and secondary amines. Unless otherwise stated, all reagents and solvents were purchased from commercial suppliers and used without further purification. Analytical thin-layer chromatography (TLC) was performed on precoated silica gel 60F254 plates, with spots visualized under UV light. 1 Dog 13C NMR spectra were recorded on a 400 MHz spectrometer using CDCl3 as the solvent and tetramethylsilane (TMS) as the internal standard. Infrared (IR) spectra were recorded on an FT-IR spectrometer. High-resolution mass spectrometry (HRMS) data for the compounds were also recorded. Column chromatography was performed using silica gel (60-120 mesh) and an ethyl acetate / petroleum ether mixture as the eluent.

[0035] Fig. Figure 3 shows a diagram illustrating the plausible mechanism for the formation of methylsulfonylacrylamidine in accordance with one embodiment of the present disclosure.

[0036] In Fig.A feasible route for the synthesis of sulfonylacrylamidine is outlined in Figure 3. The synthesis begins with the reaction of an N,N-disubstituted amine and propargyl bromide to generate N,N-disubstituted propargylamine (I). This intermediate then interacts with Cu(I) to form Cu(I) acetylide (II). Subsequently, the Cu(I) acetylide reacts with mesyl azide to form a Cu-coordinated five-membered triazole ring (III). The triazole ring undergoes rearrangement to give N-sulfonyl ketenimine. This ketenimine then undergoes a 4-exo-dig cyclization with the amine nitrogen atom, leading to the formation of a highly unstable azetidine ring (IV). Finally, an E1cB elimination mechanism facilitates ring opening to yield the desired product, methylsulfonylacrylamidine (V).

[0037] The α-glucosidase inhibitory activities of the compounds were evaluated. The assay was performed by adding 20 µl of various concentrations of compounds 4a-4h, 20 µl of α-glucosidase solution, and 135 µl of potassium phosphate buffer to a 96-well plate. The mixture was incubated at 37°C for 20 minutes. Subsequently, 25 µl of p-nitrophenyl-α-D-glucopyranoside (substrate, 4 mM) was added to the plate, followed by further incubation at 37°C for 20 minutes. The absorbance was measured at 405 nm using a microplate reader (Benesphera, Elisa Microplate Reader). The percentage inhibition was calculated using the following formula: Inhibition(%)=[(1−ΔA sample / ΔA control)× 100%], where ΔA sample and ΔA control represent the absorbance values ​​in the presence and absence of the test substances, respectively. The IC50 values ​​were determined by plotting the percentage of inhibition against the concentrations of the test substances and performing a linear regression.

[0038] The enzyme kinetic assay for α-glucosidase was performed according to the described methodology. The substrate, pNPG, and compound 4h were tested at different concentrations. The Michaelis constant (Km) and the maximum reaction rate (Vmax) were determined using the Lineweaver-Burk plot.

[0039] Molecular docking was performed to analyze the binding interactions between compound 4h and α-glucosidase. The 3D structures of compounds 4f and 4h were generated, with the search grid for α-glucosidase defined with the following parameters: center_x = 40, center_y = 40, center_z = 40, size_x = 0.334, size_y = -26.487, and size_z = 86.844. A depletion value of 10 was used. The pose with the highest docking score was selected and visualized for further analysis.

[0040] The cytotoxicity of compound 4h was assessed using the CCK-8 assay. HEK 293 cells were cultured in 96-well plates at a density of 1 × 10 -6Cells were seeded per well and incubated for 24 hours. Subsequently, the cells were treated with different concentrations of the compound for 4 hours and incubated for another 48 hours at 37°C and 5% CO2. After this time, 10 µL of CCK-8 reagent was added to the culture medium, and the cells were then incubated for 1 hour at 37°C. Absorbance was measured at 450 nm using a microplate reader (Benesphera, Elisa Microplate Reader). Relative cell viability (%) was calculated based on the absorbance values.

[0041] The ADME properties of the molecules were predicted using the Molinspiration online property toolkit. Key pharmaceutical properties such as miLogP, hydrogen bond donors, hydrogen bond acceptors, the number of rotatable bonds, molecular volume, and molecular weight were evaluated using Lipinski's rule of five.

[0042] The present invention focuses on the α-glucosidase inhibitory activity and structure-activity relationships (SARs) of the synthesized compounds 4a-4h. In an in vitro assay, these compounds exhibited varying degrees of α-glucosidase inhibition with IC50 values ​​between 10.35 ± 0.15 and 60.39 ± 1.77 µM. Compound 4h exhibited the highest inhibitory activity with an IC50 of 10.35 ± 0.15 µM, followed by compound 4f with an IC50 of 14.54 ± 0.19 µM. Acarbose, used as a positive control, had a much higher IC50 of 832.22 ± 2.00 µM. SAR analysis revealed that the introduction of electron-donating groups on the nitrogen of the secondary amine significantly increased the activity, highlighting the importance of substitution patterns in determining the biological activity of these compounds.

[0043] Enzyme kinetics analysis for compound 4h revealed that it acts as a competitive inhibitor of α-glucosidase. The Lineweaver-Burk plot showed that the slope increased with higher concentrations of 4h, but the Vmax remained unchanged, confirming competitive inhibition. Further analysis using Dixon plots estimated the Ki value to be 1.85 × 10 -6 M. This mechanism suggests that compound 4h binds only to the free enzyme, which supports previous studies.

[0044] Molecular docking provided insights into the interaction between the synthesized compounds and α-glucosidase. Using a homology model based on the crystal structure of isomaltase from Saccharomyces cerevisiae (PDB ID: 5NN5), docking analysis revealed that the compounds bind within a hydrophobic pocket of the enzyme and interact with residues such as HIS717, LEU868, and GLU869. Hydrophobic interactions, hydrogen bonds, and pi-sulfur stacking interactions were involved in the binding, supporting the inhibitory effect observed in vitro. The binding energy and conformations were described in more detail for each compound, providing valuable information for the development of more potent α-glucosidase inhibitors.

[0045] The in vitro cytotoxicity assay showed that compounds 4f and 4h exhibited low toxicity to human embryonic kidney cell lines (HEK 293), with IC50 values ​​of 14.54 ± 0.19 µM and 10.35 ± 0.15 µM, respectively. This suggests that these compounds have a good safety profile and potential as hypoglycemic agents.

[0046] The ADME predictions for compounds 4a-4h were evaluated using the Molinspiration online property calculation toolkit. The results showed excellent absorption rates of approximately 92% for all compounds. Based on Lipinski's rule of five, all compounds except 4g and 4h were predicted to be orally active. These results underscore the potential of the synthesized compounds as safe and effective candidates for further development as α-glucosidase inhibitors with hypoglycemic applications.

[0047] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will understand that one or more of the described elements may well be combined to form 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, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the acts of a flowchart need not be performed in the order shown; nor do all acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of the embodiments is in no way limited by these specific examples.Numerous variations are possible, whether explicitly stated in the description or not, such as differences in structure, dimensions, and use of materials. The scope of the embodiments is at least as broad as indicated in the following claims.

[0048] Advantages, other benefits, and solutions to problems have been described above with respect to specific embodiments. However, the advantages, benefits, solutions to problems, and components that may cause an advantage, benefit, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all of the claims. REFERENCES: 102 Reaction vessel unit 104 Reagent supply unit 106 Reaction Monitoring Unit 108 Temperature control unit 110 stirring unit 112 Characterization unit 114 Analysis Unit 116 Computational analysis unit 118 Quality Control Unit

Claims

[1] System (100) for the synthesis of novel methylsulfonylacrylimidamide analogues, comprising: a) a reaction vessel unit (102) configured to maintain a controlled reaction environment; b) a reagent supply unit (104) configured to sequentially introduce: - Propargyl bromide, - secondary amine, - Triethylamine, - Water, - Mesylazide and - copper iodide catalyst; c) a reaction monitoring unit (106) configured to: - to detect the evolution of nitrogen gas, and - to monitor the completion of the reaction by thin-layer chromatography; d) a temperature control unit (108) configured to maintain room temperature conditions during the reaction; and e) a stirring unit (110) in communication with the reaction vessel unit (102) configured to provide continuous stirring of the reaction mixture. [2] System (100) according to claim 1, further comprising a characterization unit (112) configured to perform: - Proton nuclear magnetic resonance spectroscopy at 400 MHz, - Carbon-13 nuclear magnetic resonance spectroscopy, - Fourier transform infrared spectroscopy, and - Measurements of chemical shift and coupling constant. [3] The system (100) according to claim 1 further comprises an analysis unit (114) configured to: - to measure the α-glucosidase inhibitory activity, - to determine IC50 values, and - To conduct cytotoxicity assessments against human cell lines. [4] System (100) according to claim 1, wherein the reagent dispensing unit (104) is configured to provide precise stoichiometric ratios of: - 0.5 millimoles of propargyl bromide, - 0.5 millimoles of secondary amine, - 0.6 millimoles of triethylamine, - 0.6 millimoles of mesylazide, and - 0.05 millimoles of copper iodide. [5] The system (100) of claim 1, further comprising a computation analysis unit (116) configured to: - to conduct molecular docking studies, - perform ADME predictions, and - to check compliance with Lipinski's rule of five. [6] The system (100) according to claim 1 further comprises a quality control unit (118) configured to: - to monitor the reaction progress using thin-layer chromatography, - to verify product purity, and - to validate spectroscopic data. [7] The system (100) of claim 2, wherein the characterization unit (112) is configured to use residual solvent signals as internal references for nuclear magnetic resonance measurements. [8] The system (100) of claim 3, wherein the analysis unit (114) is configured to compare inhibitory activities with standard acarbose.

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