Synthesis and In Silico Analysis of New Polyheterocyclic Molecules Derived from [1,4]-Benzoxazin-3-one and Their Inhibitory Effect against Pancreatic α-Amylase and Intestinal α-Glucosidase

This study focuses on synthesizing a new series of isoxazolinyl-1,2,3-triazolyl-[1,4]-benzoxazin-3-one derivatives 5a–5o. The synthesis method involves a double 1,3-dipolar cycloaddition reaction following a “click chemistry” approach, starting from the respective [1,4]-benzoxazin-3-ones. Additionally, the study aims to evaluate the antidiabetic potential of these newly synthesized compounds through in silico methods. This synthesis approach allows for the combination of three heterocyclic components: [1,4]-benzoxazin-3-one, 1,2,3-triazole, and isoxazoline, known for their diverse biological activities. The synthesis procedure involved a two-step process. Firstly, a 1,3-dipolar cycloaddition reaction was performed involving the propargylic moiety linked to the [1,4]-benzoxazin-3-one and the allylic azide. Secondly, a second cycloaddition reaction was conducted using the product from the first step, containing the allylic part and an oxime. The synthesized compounds were thoroughly characterized using spectroscopic methods, including 1H NMR, 13C NMR, DEPT-135, and IR. This molecular docking method revealed a promising antidiabetic potential of the synthesized compounds, particularly against two key diabetes-related enzymes: pancreatic α-amylase, with the two synthetic molecules 5a and 5o showing the highest affinity values of 9.2 and 9.1 kcal/mol, respectively, and intestinal α-glucosidase, with the two synthetic molecules 5n and 5e showing the highest affinity values of −9.9 and −9.6 kcal/mol, respectively. Indeed, the synthesized compounds have shown significant potential as antidiabetic agents, as indicated by molecular docking studies against the enzymes α-amylase and α-glucosidase. Additionally, ADME analyses have revealed that all the synthetic compounds examined in our study demonstrate high intestinal absorption, meet Lipinski’s criteria, and fall within the required range for oral bioavailability, indicating their potential suitability for oral drug development.

Isoxazoline is an innovative heterocycle that is gaining increasing interest due to its diverse pharmacological activities, making it a primary focus for several research groups worldwide.Primarily recognized for its potential in synthesizing new antibacterial agents [33], it also possesses other highly exciting biological activities, such as antidiabetic [34,35], anticancer [36], antioxidant [37], antimicrobial [38], anti-inflammatory [39], antifungal [40], antiviral [41], and anti-Alzheimer properties [42].The development of novel isoxazoline derivatives continues to be a major focus in medical research.
Diabetes mellitus is a persistent metabolic disorder marked by elevated levels of glucose in the bloodstream resulting from a dysfunction in the production or function of insulin [43].Diabetes management is based on regulating blood glucose levels as closely as possible to normal physiological levels to prevent the development of chronic diabetic complications such as retinopathy, nephropathy, and neurological and cardiovascular diseases [44].Among the strategies for treating diabetic patients is the administration of medications endowed with inhibitory effects on the enzymatic activity of the α-amylase and α-glucosidase enzymes [45,46].
All the synthesized compounds were characterized utilizing proton nuclear magnetic resonance ( 1 H NMR) and carbon-13 nuclear magnetic resonance ( 13 C NMR) spectroscopy (see the experimental section) (Table 1).We see that these 1,3-dipole cycloaddition reactions are totally regioselective because the direction of attack of the dipole is unique (Figure 2).This reaction led to the synthesis of isoxazoline, which constitutes the third heterocyclic component and also exhibits very interesting biological activities.

Computational Analysis Using Molecular Docking
Molecular docking, an advanced computational technique, is frequently utilized to offer valuable insights into the molecular mechanisms of pharmacologically active substances.In this study, molecular docking was employed to unveil the potential mechanism of action associated with the fifteen synthetic molecules' pancreatic α-amylase and intestinal α-glucosidase activities.

Computational Analysis Using Molecular Docking
Molecular docking, an advanced computational technique, is frequently utilized to offer valuable insights into the molecular mechanisms of pharmacologically active substances.In this study, molecular docking was employed to unveil the potential mechanism of action associated with the fifteen synthetic molecules' pancreatic α-amylase and intestinal α-glucosidase activities.

In Silico, Inhibitory Activity of Synthetic Molecules on α-Amylase Activity
The provided data, which comprise binding affinity values, imply that the molecule under study potentially exhibits either a heightened or diminished affinity toward the specified target in comparison with a native ligand, namely acarbose, if a decrease in binding energy correlates with an increase in compound affinity (Table 2).The active sites of pancreatic α-amylase predominantly feature amino acid residues such as Glu A:233 and Asp A:197, A:300, alongside pivotal residues like Arg A:195 and A:337; Trp A:58, A:284, A:203, and A:59; His A:101, A:201, and A:299; Phe A:298, A:265, and A:295; Asn A:298; Gly A:306; Ala A:307; and Tyr A:62 [55][56][57].Within this context, it is observed that all the examined molecules, except 5n, exhibit lower free binding energy values compared with the native ligand, suggesting potent inhibitory potential.Molecules 5a and 5o demonstrate the lowest free binding energy values, standing at 9.2 and 9.1 kcal/mol, respectively.Notably, both 5a and 5o establish electrostatic bonds with amino acid residues surrounding the protein's active site, primarily in the forms of Pi-sigma, Pi-Pi stacked, and Pi-alkyl interactions (Figure 3).Nevertheless, it is noteworthy that 5o additionally forms a conventional hydrogen bond with the amino acid residue His A:202.The findings of the computational analysis indicate that the observed antihyperglycemic effects and inhibition of pancreatic α-amylase can be ascribed to these molecules.
electrostatic bonds with amino acid residues surrounding the protein's active site, primarily in the forms of Pi-sigma, Pi-Pi stacked, and Pi-alkyl interactions (Figure 3).Nevertheless, it is noteworthy that 5o additionally forms a conventional hydrogen bond with the amino acid residue His A:202.The findings of the computational analysis indicate that the observed antihyperglycemic effects and inhibition of pancreatic α-amylase can be ascribed to these molecules.

In Silico, Inhibitory Activity of Synthetic Molecules on α-Glucosidase Activity
The data presented, in the form of binding affinity values, suggest that the molecule under investigation may exhibit either a heightened or diminished affinity for the specified target in comparison with the native ligand (acarbose), assuming a decrease in binding energy correlates with an increase in the compound's affinity (Table 3).

In Silico, Inhibitory Activity of Synthetic Molecules on α-Glucosidase Activity
The data presented, in the form of binding affinity values, suggest that the molecule under investigation may exhibit either a heightened or diminished affinity for the specified target in comparison with the native ligand (acarbose), assuming a decrease in binding energy correlates with an increase in the compound's affinity (Table 3).The active sites of α-glucosidase are primarily surrounded by the amino acid residues Trp A:376, Asp A:404, Leu A:405, Ile A:441, Trp A:481, Asp A:518, Met A:519, Arg A:600, Trp A:613, Asp A:616, Phe A:649, and His A:674 [58].
Our observations within this framework indicate that all examined molecules exhibit a significant free binding energy compared with the native ligand, ranging from −8.8 to −9.6 kcal/mol (Table 2).Specifically, compounds 5e and 5n display the lowest values of free binding energy, at −9.9 and −9.6 kcal/mol, respectively.It is noteworthy that these molecules establish hydrogen bonds (interactions between a hydrogen atom bonded to an electronegative atom and a neighboring electronegative atom) and electrostatic bonds (interactions between oppositely charged entities) with the amino acid residues surrounding the protein's active site, primarily in the forms of conventional hydrogen bonds, Pi-sigma interactions (bonds between a pi electron and a sigma atom), Pi-Pi stacked interactions (interactions between pi systems), and Pi-alkyl interactions (interactions between a pi system and alkyl groups).Specifically, compound 5n forms four conventional hydrogen bonds with the amino acid residues Tyr A:360, Met A:363, Arg A:608, and Glu A:866 (Figure 4A), while compound 5e forms five hydrogen bonds with Tyr A:360, Met A:363, His A:584, Arg A:608, and Glu A:866 (Figure 4B) from the active site of α-glucosidase.The computational findings suggest that these molecules may contribute to the observed antihyperglycemic effects and the inhibition of pancreatic α-glucosidase, underscoring the significance of hydrogen bonding interactions in modulating enzymatic activity.

ADME Analysis
In silico ADME studies are essential to advancing pharmaceutical development by offering a cost-effective method for predicting how a drug will act within the body [59].Utilizing computer models for early pharmacokinetic assessment, these studies enable the swift selection of drug candidates and streamline development processes.They help minimize the risk of adverse side effects, decrease the likelihood of drug development failures, and increase the chances of clinical success, making them an invaluable tool in The computational findings suggest that these molecules may contribute to the observed antihyperglycemic effects and the inhibition of pancreatic α-glucosidase, underscoring the significance of hydrogen bonding interactions in modulating enzymatic activity.

ADME Analysis
In silico ADME studies are essential to advancing pharmaceutical development by offering a cost-effective method for predicting how a drug will act within the body [59].
Utilizing computer models for early pharmacokinetic assessment, these studies enable the swift selection of drug candidates and streamline development processes.They help minimize the risk of adverse side effects, decrease the likelihood of drug development failures, and increase the chances of clinical success, making them an invaluable tool in modern drug discovery and development [60].In order to comply with Lipinski's rule of five and Veber's rule, compounds deemed suitable for oral drug development should typically not exceed one violation of the following criteria: (1) no more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms), (2) an octanol-water partition coefficient log P (MLogP) < 5, (3) a molecular mass < 500 daltons, and (4) no more than 5 hydrogen bond donors [61].In our study, we observed that all the compounds examined met Lipinski's criteria, indicating their potential suitability for oral drug development.
The blood-brain barrier (BBB) serves as a crucial barrier between the systemic circulation and the central nervous system, protecting the brain through both biochemical processes, like enzyme reactions, and physical mechanisms such as active expulsion systems [62].Our investigation revealed that synthetic molecules are unable to cross this barrier, because they have a TPSA > 79 Å 2 , as shown in Table 4 and illustrated in the yellow portion of Figure 5.No molecules disperse in this yellow area.Additionally, most of the compounds analyzed were determined to be P-glycoprotein non-substrates (PGP-), except for 5f, 5l, 5j, 5n, 5i, and 5d, which were determined to be PGP+ (Figure 4).A molecule that is strongly absorbed by the intestine offers significant advantages in terms of bioavailability, efficacy, convenience, and tolerance, making it a promising candidate for the development of oral medications [63].Notably, all the synthetic compounds examined in our study demonstrate high intestinal absorption.Cytochrome P450 is a crucial enzyme for detoxification, primarily located in the liver [64].Our analysis identified that the majority of the compounds are neither inhibitors nor substrates of CYP450 enzymes, particularly CYP1A2, except 5i, 5m, 5g, 5a, 5e, and 5k, which were identified as CYP1A2 inhibitors (Table 3).This finding implies a lower likelihood of medication metabolism disruption, thereby strengthening the safety profile of the synthetic compounds.Figure 6 displays the bioavailability profiles of these drugs.The pink zone in these radar graphs corresponds to the oral bioavailability space.A chemical's characteristics must entirely fall within this specified region to qualify as drug-like [65].In the present study, all the synthetic compounds meet the required range for oral bioavailability, suggesting their potential as drug candidates.

General
Merck-60 silica gel (230-400 mesh E) was employed for column chromatography.Melting points for compounds 3 and 5a-5o were measured using a Kofler bench (FST, Beni Mellal, Morocco).Reaction progress was tracked with thin-layer chromatography (TLC) on aluminum plates coated with silica gel 60 F254 (E.Merck).Nuclear magnetic resonance (NMR) spectra were obtained on a Varian Unity Plus spectrometer (CNRST, Rabat, Morocco)at 500 MHz for 1 H NMR and at 125.76 MHz for 13 C NMR.Chemical shifts were given in parts per million (ppm), with coupling constants (J) noted in Hertz (Hz).The signals were characterized as s (singlet), d (doublet), t (triplet), and m (multiplet), and tetramethylsilane Si(CH ) was used as the reference.

General
Merck-60 silica gel (230-400 mesh E) was employed for column chromatography.Melting points for compounds 3 and 5a-5o were measured using a Kofler bench (FST, Beni Mellal, Morocco).Reaction progress was tracked with thin-layer chromatography (TLC) on aluminum plates coated with silica gel 60 F254 (E.Merck).Nuclear magnetic resonance (NMR) spectra were obtained on a Varian Unity Plus spectrometer (CNRST, Rabat, Morocco)at 500 MHz for 1 H NMR and at 125.76 MHz for 13 C NMR.Chemical shifts were given in parts per million (ppm), with coupling constants (J) noted in Hertz (Hz).The signals were characterized as s (singlet), d (doublet), t (triplet), and m (multiplet), and tetramethylsilane Si(CH 3 ) 4 was used as the reference.
3.2.Procedure for the Preparation of Compound 3 by "Click Chemistry" (CuAAC) A solution of 1 mmol of compound 1 and 2 mmol of 3-azidoprop-1-ene 2 in 8 mL of methanol was prepared, to which 1 mmol of sodium ascorbate and 1 mmol of CuSO 4 •5H 2 O dissolved in 7 mL of distilled water were added.The reaction mixture was stirred at room temperature for 3 h and monitored using TLC.Following filtration and concentration under decreased pressure, the resultant substance was then submitted to column chromatography on silica gel, utilizing a mixture of ethyl acetate and hexane at a ratio of 3 to 7 as the eluting solvent.Compound 3 was obtained with a good yield of 81%.

Molecular Docking Analysis
The molecular docking analysis was conducted following the guidelines outlined in the reference [66][67][68].The crystalline structures of α-amylase (PDB ID: 1SMD) and α-glycosidase (PDB ID: 5NN5) were obtained from the RCSB protein database (http: //www.rcsb.org/pdb)(accessed on 3 March 2024), established at the Brookhaven National Laboratory in 1971.The removal of water molecules was achieved using AutoDock Tools v1.5.7, while also incorporating polar hydrogens and Kollman charges; co-crystallized ligands were excluded; and the protein was saved in the "pdbqt" format.The two-dimensional configuration of each ligand was converted to the three-dimensional configuration using Avogadro version 1.2.0 software, as depicted in [69,70].Using AutoDock Tools (version 1.5.6), the final pdbqt file of the ligand was obtained.The grid box representing the docking search space was enlarged to better fit the active binding site.The coordinates of the grid box for the two enzymes, α-amylase and α-glycosidase, were defined as follows: for α-amylase, the centers (x, y, and z) were set at 8.349, 58.705, and 19.096, while for α-glucosidase, the centers (x, y, and z) were fixed at 1.591, −26.522, and 87.364, with a uniform grid box size maintained at 40.The results for the docked ligand complexes were expressed as ∆G binding energy values in kcal/mol.Acarbose, an agent with a history of 30 years in treating type 2 diabetes, is utilized to prevent postprandial hyperglycemia by blocking carbohydrate digestion in the small intestine.In this computational section of the investigation, acarbose was employed as the native ligand.The process of generating 2D molecular interaction diagrams and examining protein-ligand binding interactions was carried out using Discovery Studio 4.1 (Dassault Systems Biovia, San Diego, CA, USA).

ADME Studies
Understanding pharmacokinetic properties, including absorption, distribution, metabolism, and excretion (ADME), is essential for comprehending how a substance acts in the body [71].These stages describe the journey of a substance from absorption to elimination.Computational tools have become indispensable for predicting the ADME characteristics of molecules, assessing their ability to cross cellular barriers and interact with essential transporters and enzymes for absorption and excretion, and determining their metabolic stability [59].In our approach to molecule evaluation, we have chosen to use the Swis-sADME platform (available online: www.swissadme.ch,accessed on 10 April 2024) [61].This platform allows us to thoroughly examine the physicochemical attributes of synthetic molecules, their potential as therapeutic agents, and their pharmacokinetic properties, thus providing a comprehensive understanding of their ADME profile [72].

Figure 3 .
Figure 3. Two-dimensional schemes of the interactions with the amino acid residues of the two potent synthetic molecules, (A) 5a, (B) 5o, and the native ligand, Acarbose (C).

Figure 3 .
Figure 3. Two-dimensional schemes of the interactions with the amino acid residues of the two potent synthetic molecules, (A) 5a, (B) 5o, and the native ligand, Acarbose (C).

Figure 4 .
Figure 4. Two-dimensional schemes of the interactions with the amino acid residues of the two potent synthetic molecules, (A) 5n, (B) 5e, and the native ligand, Acarbose (C).

Figure 4 .
Figure 4. Two-dimensional schemes of the interactions with the amino acid residues of the two potent synthetic molecules, (A) 5n, (B) 5e, and the native ligand, Acarbose (C).

Figure 6 .
Figure 6.Bioavailability radar of synthetic molecules.The pink region corresponds to the ideal range for each characteristic in terms of oral bioavailability (Lipophilicity, solubility, molecular weight, saturation and flexibility).

Figure 6 .
Figure 6.Bioavailability radar of synthetic molecules.The pink region corresponds to the ideal range for each characteristic in terms of oral bioavailability (Lipophilicity, solubility, molecular weight, saturation and flexibility).

Table 2 .
H-bonds, binding energy, and interacting amino acids of phytocompounds found in the synthetic molecules 5a-5o target protein.
1 Acarbose, a native ligand of α-amylase; * The potent ligands in comparison with the native ligand.

Table 3 .
H-bonds, binding energy, and interacting amino acids of phytocompounds found in the synthetic molecules 5a-5o target protein.

Table 4 .
Evaluation of the pharmacokinetic properties (ADME) of the Synthetic Compounds.