Front. Chem. Frontiers in Chemistry Front. Chem. 2296-2646 Frontiers Media S.A. 1740409 10.3389/fchem.2025.1740409 Original Research Regioselective synthesis of novel spiro-isoxazolines congeners as antimicrobial agents: in vitro and in-silico assessments Bouzammit et al. 10.3389/fchem.2025.1740409 Bouzammit Rachid 1 Conceptualization Investigation Resources Writing - original draft Ait Assou Soumia 2 Investigation Resources Writing - original draft Er-Rajy Mohammed 3 Formal Analysis Software Writing - original draft Aflak Noura 4 Data curation Software Writing - original draft Bahsis Lahoucine 5 Writing - original draft Data curation Formal Analysis Chalkha Mohammed 1 6 * Investigation Validation Writing - review and editing El Hassouni Mohammed 2 Validation Visualization Writing - review and editing Lachkar Mohammed 1 Resources Validation Writing - review and editing Ben Hadda Taibi 7 Investigation Validation Writing - original draft Benson Daryn 7 Validation Visualization Writing - original draft Alyousef Abdullah A. 8 Funding acquisition Validation Writing - original draft Aboul-Soud Mourad A. M. 9 Funding acquisition Investigation Writing - review and editing Giesy John P. 10 11 12 Formal Analysis Funding acquisition Writing - review and editing Al Houari Ghali 1 * Conceptualization Project administration Supervision Writing - review and editing Engineering Laboratory of Organometallic, Molecular Materials and Environment (LIMOME), Faculty of Sciences, University Sidi Mohamed Ben Abdellah, Fez, Morocco Biotechnology, Environment, Agri-Food and Health Laboratory, Faculty of Sciences Dhar El Mahraz, Sidi Mohamed Ben Abdellah University, Fez, Morocco LIMAS Laboratory, Chemistry Department, Faculty of Sciences Dhar El Mahraz, Sidi Mohamed Ben Abdellah University, Fez, Morocco Team of Organic Chemistry and Valorization of Natural Substances, Faculty of Sciences, University Ibn Zohr, Agadir, Morocco Laboratory of Analytical and Molecular Chemistry/LCAM, Polydisciplinary Faculty of Safi, University Cadi Ayyad, Safi, Morocco Laboratory of Materials Engineering for the Environment and Natural Resources, Faculty of Sciences and Techniques, University of Moulay Ismail, Errachidia, Morocco Euro-Medeterranean University of Fes (UEMF), Fez, Morocco Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia Center of Excellence in Biotechnology Research (CEBR), College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia Department of Veterinary Biomedical Sciences and Toxicology Centre, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada Department of Integrative Biology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, United States Department of Environmental Sciences, Baylor University, Waco, TX, United States Correspondence: Mohammed Chalkha, mohammed.chalkha1@usmba.ac.ma; Ghali Al Houari, ghalialhouari@gmail.com 29 12 2025 2025 13 1740409 05 11 2025 27 11 2025 27 11 2025 Copyright © 2025 Bouzammit, Ait Assou, Er-Rajy, Aflak, Bahsis, Chalkha, El Hassouni, Lachkar, Ben Hadda, Benson, Alyousef, Aboul-Soud, Giesy and Al Houari. 2025 Bouzammit, Ait Assou, Er-Rajy, Aflak, Bahsis, Chalkha, El Hassouni, Lachkar, Ben Hadda, Benson, Alyousef, Aboul-Soud, Giesy and Al Houari https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction

A new class of spiroisoxazolines was efficiently synthesized through a regioselective cycloaddition between arylidene tetralone 1 and arylnitrile oxides 2, characterized and assessed for their in vitro antimicrobial activity.

 Methods

The structures and regioselectivity of the obtained cycloadducts were confirmed by 1H, 13C-NMR, IR, elemental analysis, and mass spectrometry, and further supported by theoretical calculations that explained the reaction process and the regioselective results. The antimicrobial profile of the synthetized spiro derivatives was evaluated against the yeast Candida albicans, the Gram-postive bacteria (Staphylococcus aureus and Bacillus subtilis), and the Gram-negative bacteria (Escherichia coli and Pectobacterium basiliensis). In addition, in silico studies were carried out to rationalize the experimental findings and provide mechanistic insight.

 Results and Discussion

Two spiroisoxazolines, defined as 3b and c, showed notable antimicrobial activity, producing inhibition zones between 8.33 ± 0.57 and 14.00 ± 2.00 mm. Compound 3b was active against all tested strains and demonstrated ampicillin-comparable MIC values of 10 μg/mL against E. coli, P. brasiliensis, and B. subtilis. It showed moderate to weak activity against S. aureus (90 μg/mL) and C. albicans (300 μg/mL). Compound 3c displayed selective activity toward Gram-positive bacteria with MIC values of 50 and 500 μg/mL against B. subtilis and S. aureus, respectively. Molecular docking studies confirmed the high binding affinities of 3b and 3c toward the active sites of the targeted proteins, in agreement with the antimicrobial results. POM analyses further indicated the coexistence of antifungal (O1δ−—O2δ−) and antiviral (O1δ−—N1δ−) pharmacophoric sites, although steric constraints introduced by two methyl substituents may limit their optimal interaction. The calculations also confirmed favorable bioavailability and the absence of predicted toxicity for all compounds. Overall, this combined experimental -theoretical study highlights the mechanistic basis and biological relevance of these spiroisoxazolines, underscoring their potential as promising scaffolds for the rational design of antiviral drug candidates.

 1,3-dipolar cycloaddition regioselectivity spiroisoxazoline antimicrobial activity in silico studies reactions with azo Schiff bases Petra/Osiris/Molinspiration analyses The authors declare that financial support was received for the research and/or publication of this article. This study was financially supported by Ongoing Research Funding Program, (ORF-2025-1175), King Saud University, Riyadh, Saudi Arabia. section-in-acceptance Organic Chemistry Introduction

Heterocycles play a fundamental role in organic chemistry, pharmaceuticals, biology, and materials science. They constitute the core framework of a wide range of compounds with significant chemical, biological, pharmacological, and industrial applications (Mezgebe and Mulugeta, 2022; Uppadhayay et al., 2022). Their structural versatility and tunable reactivity make them indispensable to modern chemistry. Representing nearly two-thirds of all known organic compounds, heterocycles are crucial in the discovery and development of bioactive molecules (Cao et al., 2017).

Due to their pharmacological (Shinde et al., 2024), medicinal (Wang et al., 2023), and industrial (Guo et al., 2019) potential, characterized by their exceptional reactivity and their ability to rapidly develop biologically active compounds, as a class of heterocycles, isooxazoline derivatives have attracted significant interest from chemists. This has led to a focus on the synthesis of new isooxazoline compounds to enhance their biological efficacy (Chalkha et al., 2022). Among the methods for the synthesis of isoxazoline compounds, 1,3-dipolar cycloaddition (1,3-DC) reactions are particularly noteworthy. Although other methods exist (Paciorek et al., 2022), these reactions represent the most versatile, widely used, and straightforward means of obtaining five-membered heterocyclic compounds containing oxygen and nitrogen atoms (Rana and Ansari, 2023).

The spiro function attached to a single carbon is a common structural motif found in many compounds with significant biological activity (Hong and Wang, 2013), such as anticancer (Aljohani et al., 2022), antitumor (Das et al., 2019; Najim et al., 2010), anti-tubercular (Mane et al., 2021), anti-inflammatory (Afsar et al., 2024), antifungal (Sawhney et al., 2022), and antiviral (Das et al., 2020). Due to their wide range of potential pharmacological applications, spiroisoxazolines are particularly important spiro-heterocyclic compounds in organic synthesis (Kumar et al., 2024). Some spiroisoxazoline compounds, such as 11-deoxyfistularin-3 (Ferreira Montenegro et al., 2024), and fluoro-substituted spiro-isooxazolines (Das et al., 2020), have demonstrated cytotoxic activity against cancer. Other Spiro compounds of this family, including aplysinamisin-1 (Rani et al., 2021), agelorin A, B (Moriou et al., 2021), derivatives of (R)–carvone (Dai et al., 2016), derivatives of (−)-α-santonin (Kaur et al., 2017), derivatives of artemisinin (Pratap et al., 2019) and Aerophobin-1 (Carnovali et al., 2022), have shown Antimicrobial (Raju et al., 2024), anticancer (Kalhor et al., 2024), antioxidants (AL-Adhreai et al., 2022) or antiproliferative properties (Alminderej et al., 2023) (Figure 1). However, despite these advances, previous studies still present several limitations. Many reported synthetic procedures suffer from limited regioselectivity, narrow substrate scope, or require harsh conditions. In addition, several spiroisoxazoline derivatives exhibit only moderate antimicrobial activity (Madadi Mahani et al., 2025), and very few studies have explored the incorporation of tetralone units into spiroisoxazoline frameworks. Moreover, mechanistic understanding remains incomplete, as only a limited number of publications combine experimental synthesis with computational approaches such as DFT, molecular docking, or POM theory. These gaps highlight the need for new spiroisoxazoline derivatives supported by a deeper mechanistic and biological analysis.

Biologically active natural products containing spiroisoxazoline structures.

Chemical structures of four compounds: 11-Deoxyfistularin-3, Aerophobin-1, Amplysinamisin I, and Agelorin A and B. Each contains a red-highlighted oxime group, and brominated aromatic rings. Structures are labeled with their respective compound names.

Due to the outstanding biological properties of isooxazoline units and spiro heterocyclic compounds (Raju et al., 2024), and consistent with our ongoing research efforts, focused on the synthesis of new heterocyclic systems intended for therapeutic use (Bouzammit et al., 2024a; Bouzammit et al., 2024b; Bouzammit et al., 2024c; Bouzammit et al. 2025; Ech-chihbi et al., 2025; Kanzouai et al., 2023; Wang et al., 2021), the main objective of the current study was to synthesize novel spiro heterocyclic derivatives containing isooxazoline and (Bouzammit et al., 2025) tetralone units. The resulting spiroisoxazolines were subsequently evaluated for their potential in vitro antibacterial activity against specific pathogenic microbial strains. In addition, in silico studies including ADME-T predictions, molecular docking simulations, and POM analysis were performed to support and explain the experimental results obtained.

 Materials and methods

The Supplementary Material file provides a complete description of the general information, including solvents, reagents, and instruments, employed during the syntheses of spiroisoxazolines, and characterization of each compound.

Computational methods

All optimized molecular structures were calculated through the program “Gaussian 09” (Frisch et al., 2009) and use of the B3LYP/6-31G (d,p) basis set (Lee et al., 1988). Chloroform was used as the solvent in the polarizable continuum model (COCM). Indexes of chemical hardness (µ)/electronic chemical potential (η) were calculated (Equations 1, 2). η = E LU − E HO µ = E HO + E LU / 2 Where LU is LUMO and HO is HOMO. The expressions of global nucleophilicity (N) and electrophilicity (ω) indexes were calculated (Equations 3, 4) N = E HO   Nu − E HO   TCE

ω = µ2/2η (Chattaraj et al., 2006).

Local nucleophilic (Pk-) and electrophilic (Pk+) indices were found by the obtained values of the Mulliken atomic spin density of each reagent (Chattaraj et al., 2006). Consequently, the redefinition of the local nucleophilicity (N k) and electrophilicity (ωk) indices were defined (Equations 5, 6) (Domingo et al., 2013). N k = N · P k ω k = ω · P k +

The ELF study was conducted using the Multiwfn software (Lu and Chen, 2012).

 <italic>In vitro</italic> antimicrobial assay Agar-well diffusion method

The antimicrobial activity of spiroisoxazolines was evaluated against various microbial strains, including Gram+ bacteria (Staphylococcus aureus ATCC 29213 and Bacillus subtilis ATCC 6633), Gram− bacteria (Escherichia coli K12 and Pectobacterium brasiliensis 13471), and the yeast Candida albicans ATCC 10231 through the agar-well diffusion technique. The working solution of each compound was prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a concentration of 25 mg/mL. For the antimicrobial test, tested strains were grown in Muller Hinton (MH) broth for bacteria and in Sabouraud broth for C. albicans. Subsequently, a culture equivalent to 0.5 McFarland was employed. A total of 100 μL of this culture was combined with 5 mL of MH soft agar medium (0.5% (w/v) agar) for bacteria or with 5 mL of Sabouraud soft agar (0.5% (w/v) agar) for C. albicans. This microbial suspension was then spread evenly onto the surface of either MH agar or Sabouraud agar, based on the microorganism being tested (Hockett and Baltrus, 2017). Once the microbial overlay had set, a sterile tip was used to create a 6 mm diameter hole, into which 100 µL of the working solution, was added. The Petri dishes were incubated for 24 h, either at 37 °C for bacteria or at 30 °C for C. albicans. Wells that had ampicillin (2.5 mg) and amphotericine B (2.5 mg) served as positive controls for bacterial strains and C. albicans, respectively, while DMSO was used as the negative control. Inhibitory effect was assessed by measuring the inhibition zone diameter (IZD) around the growth. The experiment was conducted in triplicate, and the mean IZD value was determined.

 Minimum inhibitory concentration (MIC)

The MIC was evaluated by employing the microdilution method, adhering to the guidelines outlined in (Pfaller et al., 2002) and the protocol described in (Ait Assou et al., 2024). In a 96-well microplate, each well was filled with the appropriate culture medium, a suitable test concentration, and about 105 cells of the tested bacteria or 103 cells of C. albicans. Stock solutions were prepared for compounds 3 and 4 with concentrations of 25000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 700, 500, 400, 300, 200, 100, 50, and 25 μg/mL. To achieve the required concentration of 2500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 70, 50, 40, 30, 20, 10, 5, and 2.5 μg/mL, 10 μL of the stock solution was added to each well, followed by the addition of the inoculum. Well 11, containing culture medium and inoculum, and well 12, containing only culture medium, served as the positive and negative controls for growth, respectively. The microplates were incubated at either 37 °C for bacteria or 30 °C for C. albicans for a duration of 24–48 h. After this incubation period, 20 μL of a 0.01% resazurin solution was introduced into each well, and the microplate was returned to the incubator at 30 °C for an additional 3 h to check the results. The growth of the microbial strains was indicated by a pink color, and the MIC was determined as the least concentration of the compound that did not result in pink color.

 Molecular docking study

Molecular docking was used to determine the ligand-receptor interaction mechanisms involved in the complex (Er-rajy et al., 2025). Compounds 3b and 3c were synthesized and designed using ChemDraw 3D 16.0, and their geometry was optimized using the MM2 method (Mills, 2006). Discovery Studio 2021 software was then used to analyze the interactions between ligands and the protein receptor, removing water molecules, correcting missing side-chain residues and fusing non-polar hydrogens (Barghady et al., 2024; Systèmes, 2024). After preparing the protein and ligand, AutoDock Tools software was used to perform the molecular docking (Trott and Olson, 2010). Lamarck’s genetic algorithm was employed to perform the docking studies, aiming to obtain the lowest binding free energy (Er-Rajy et al., 2024). In AutoDockTools, preparation of the complex involved adding polar hydrogens and Kollman charges to the protein, generating Gasteiger charges for the ligand after automatic definition of the rotating bonds, and defining a grid that covered the entire protein surface before saving both structures in PDBQT format, in order to perform blind molecular docking (Er-rajy et al., 2023). A total of 30 solutions were calculated in each case, employing a population size of 300. Based on the results of the biological assays, the following receptors were selected. In the antibacterial study, the S. aureus receptor, obtained from the Protein Data Bank (PDB ID: 3VSL), with a resolution of 2.40 Å (Yoshida et al., 2012) was used. A grid was created using the following parameters: X = 10.09, Y = −48.570, and Z = 24.870 Å. In the study of the second antibacterial activity, we used the B. subtilis receptor, obtained from the Protein Data Bank (PDB ID: 1OF0), with a resolution of 2.45 Å (Martins et al., 2002). For protein-ligand docking studies, a grid was created using the following parameters: X = −3.173 Å, Y = 33.746 Å, and Z = 42.253 Å.

 Petra/osiris/molinspiration (POM) theory

To determine the physico-chemical factors controlling the bioactivity of potential medications such as antibacterial, antifungal, antiviral and anticancer pharmacophore sites, a mixed computational Petra/Osiris/Molinspiration (POM) based model of which all three classes are available online and are free of charge, was used.

 Results and discussion Synthesis of the spiroisoxazolines

The dipolarophile 1 was prepared following the procedure described in our previously published work (Bouzammit et al., 2024c). Moreover, the dipoles 2 utilized in this study were synthesized by converting different aromatic aldehydes to their corresponding aldoximes (anti and syn), followed by a reaction with N-chlorosuccinimide in dimethylformamide (DMF) (Grundmann and Richter, 1968). Then, the obtained nitrile oxides 2 and (E)-2-ethylidene-3-methyl-3,4-dihydronaphthalene-1(2H)-one 1 were reacted in basic medium at room temperature, resulting in the diasterio- and regioselective formation of spirocycloadducts containing isoxazoline and tetralone units 3 (Scheme 1). The structures of the obtained cycloadducts are characterized by spectroscopic techniques and validated by mass spectrometry.

Spiro-isoxazoline synthesis from arylidene tetralone.

Chemical reaction scheme depicting the synthesis of compounds 3a-d and 3'a-d. Compound 1 reacts with compounds 2a-d in the presence of triethylamine (Et₃N) and dichloromethane (CHCl₃). The aromatic group Ar varies as C₆H₅, p(Cl)C₆H₄, p(OCH₃)C₆H₄, and p(CH₃)C₆H₄ in different reactions, leading to different products. Arrows indicate the flow of the reaction, resulting in two different possible reaction pathways.

The analysis of FT-IR spectra of the synthesized spiroisoxazolines shows an absorption band between 1690 and 1700 cm-1, characteristic of the carbonyl (C=O) functional group. The assignment of the different signals in the 1H NMR spectra of the four compounds 3a-d suggested the presence of two methyl groups (CH3 (3″) ) and CH3 (4″) ) appearing as successive doublets at 0.98 and 1.30 ppm, respectively. A multiplet at about 2.9 ppm corresponds to the proton H3, while the two chemically non-equivalent CH2 protons appear as two doublets of doublets located at about 2.7 and 3.7 ppm. Additionally, a quadruplet attributed to the proton H 4’ of the isooxazoline ring appeared around 4.30 ppm, confirming the regiochemistry of compound 3. These findings align well with the majority of the cycloadducts reported in the literature. In the case of the Regio-isomer 3′, we would expect higher values above 6 ppm for the proton H 5’ under the attractive effect of oxygen (Fihi et al., 1995). Compounds 3c and 3d show two distinct signals at 3.96 ppm and 2.41 ppm, respectively, indicative of the two methyl groups (Ar-OCH3) and (Ar-CH3). The regiochemistry of the resulted cycloadduct 3 is confirmed by interpreting data obtained from 13C NMR spectra. The attractive effect of oxygen is responsible for the chemical shifts observed for the spiranic carbon C 2,5' , which are around 90 ppm (Akhazzanea et al., 2011). In contrast, the structure 3′would predict much lower values for the spiranic carbon C 2,4' , which is around 60 ppm (Fihi et al., 1995). The mass spectrometry data obtained are perfectly coherent with the proposed structures. The structure of the obtained products is validated using high-resolution mass spectrometry. All mass spectra of the synthesized spiroisoxazolines 3a-d show a molecular ion peak [M + H]+ that corresponds exactly to the molecular mass of the proposed structure.

Overall, the 1,3-DC reaction of arylnitrile oxides with ethylenic dipolarophiles leads regioselectivity to 3,4-disubstituted isooxazolines 3 (Schemes 1 and 2) (Tóth et al., 1999). Additionally, the reaction proceeded in a diastereoselective manner, with the anti-approach being favored due to steric hindrance caused by the substituent CH3 at position 3 of the arylidene (Scheme 2) (Tóth et al., 1999).

Regio- and diastereoselective formation of the spiroisoxazolines.

Chemical reaction diagram showing the transformation of a cyclic ketone and an oxime chloride in the presence of triethylamine and chloroform at room temperature. The reaction proceeds via an anti approach, forming an oxaziridine derivative with an aryl group substituent. The methyl groups are highlighted in pink, and the oxime nitrogen and chlorine are shown in blue. Mechanistic study

To explain the regioselectivity observed experimentally in this 1,3-DC reaction, ELF topological and MEDT analyses were carried out for theoretical studies. The 1,3-DC reaction, also known as 32CA, has gained acknowledgment as a remarkably efficient approach for producing a wide range of organic compounds with various practical uses (Ukaji and Soeta, 2014). Recent theoretical studies have confirmed the efficiency of this cycloaddition reaction, correlating it with the electronic structures of the three-atom components (TACs) involved in the [3 + 2] cycloaddition process (Ríos-Gutiérrez et al., 2021). In this work, the 1,3-DC reaction between 2H-2-ethylidene-3-methyl-3,4-dihydronaphthalen-1-one 1 and nitrile oxide 2 leads to the construction of the corresponding isooxazoline compound by two plausible paths concerning the regioselective attacks (Scheme 3) (Tu et al., 2022).

Plausible pathways for 1,3-DC reaction between nitrile oxide 2 and 2H-2-ethylidene-3-methyl-3,4-dihydronaphthalen-1-one 1.

Chemical reaction diagram showing two pathways, A and B, for the reaction between compounds 1 and 2. Pathway A has transition states TS1 and TS2, leading to products 3a and 3b. Pathway B has transition states TS3 and TS4, leading to products 3'a and 3'b. Each product shows structural changes indicated by the pathways.

Recently, a theoretical investigation known as Molecular Electron Density Theory (MEDT) (Domingo, 2016) has been proposed to establish a robust link between three-atom components (TACs) and their interactions with ethylene compounds in 1,3-DC reactions (Ríos-Gutiérrez and Domingo, 2019b). To explain the observed regioselectivity in nitrile oxide and 2H-2-ethylidene-3-methyl-3,4-dihydronaphthalen-1-one, the MEDT approach was utilized, and the results were presented in four main sections. First, the reagents were analyzed using ELF topological analysis. Second, reactivity indices were examined using Conceptual Density Functional Theory (CDFT). Third, potential reaction profiles for the 1,3-DC reaction were investigated. Finally, ELF topological analysis was conducted on both reagents to reveal their ionic character.

ELF study of reagents

The electronic nature of starting materials has been reported to significantly affect reaction pathways and the energetic barriers (Bahsis et al., 2020). Here, ELF functions were performed to analyze the electron density distribution and elucidate the chemical structures of reagents (Figure 2) (Becke and Edgecombe, 1990). To investigate the electronic features of the cycloaddition reaction between reagents 1 and 2, an ELF analysis was performed on their optimized geometries (Figure 2). The ELF analysis of the optimized structures revealed two disynaptic basins associated with the C1-C2 bond in reagent 1. These basins account for an electron population of 3.51 e, consistent with a typical carbon–carbon double bond. In compound 2, the ELF topology shows two disynaptic basins along the C3≡N4 bond, containing a total of 6.00 electrons-indicative of a triple bond character. Additionally, a disynaptic basin on the N4-O5 bond integrates 1.52 e, and three monosynaptic basins are observed on the oxygen atom (O5), summing to 5.68 e. These observations support the characterization of the C3≡N4 linkage as a triple bond, the N4-O5 connection as a single bond, and the presence of three lone pairs on O5 (Domingo and Ríos-Gutiérrez, 2017). Overall, the results confirm that nitrile oxide derivatives act as zwitterionic 1,3-dipolar species in polar cycloaddition reactions (Figure 2) (Ríos-Gutiérrez and Domingo, 2019a).

Basin attractors and corresponding isosurfaces for both reagents of ELF analysis.

Molecular models labeled 1 and 2 are shown with electron density representations. Model 1 features attractors labeled C1 and C2 with a total V of 3.51 electrons. Model 2 displays attractors at C3, N4, O5, with V values of 6.00, 5.68, and 1.52 electrons, respectively. Green lobes represent electron densities, while red arrows highlight attractor positions. CDFT indices analysis of reagents

To explore the chemo- and regioselectivity of the reactions, global reactivity descriptors were applied, focusing on both the reactive sites and the nature of the interaction. This analysis was carried out within the conceptual DFT, including global electron density transfer (GEDT) as a key parameter (Domingo et al., 2022). The calculated global indices for both reactants are summarized in Table 1. According to this analysis, the nitrile oxide derivative 2 exhibits a higher electronic chemical potential (µ = −3.85 eV) compared to the 2H-2-ethylidene-3-methyl-3,4-dihydronaphthalen-1-one compound 1, which has a µ of −4.24 eV. This energy difference suggests that during the transition state, electron density is moved from the nitrile oxide 2 toward the dihydronaphthalenone 1. The computed nucleophilicity and electrophilicity values (Table 1) reveal that compound 2 behaves as a moderate nucleophile (N = 2.41 eV) and a moderately strong electrophile (ω = 1.45 eV). In contrast, compound 1 displayed moderate nucleophilicity (N = 2.16 eV) and a higher electrophilicity (ω = 1.86 eV), based on established reactivity scales (Domingo et al., 2002). These results indicate that 2H-2-ethylidene-3-methyl-3,4-dihydronaphthalen-1-one 1 acts predominantly as an electrophilic species in the 1,3-DC reaction, while the nitrile oxide 2 functions as the nucleophilic counterpart, indicating a polar reaction profile.

Property and reactivity indices on a global scale. All measurements are expressed in electron volts (eV).

​ EHOMO ELUMO η µ ω N
Dipolarophile 1 −6.66 −1.82 −4.24 4.84 1.86 2.16
Dipole 2 −6.41 −1.29 −3.85 5.13 1.45 2.41

Recent analyses of local reactivity indices derived from Parr functions have revealed a strong correlation between bond formation in polar reactions and the experimentally observed chemo- and regioselectivity (Domingo, 2014). The study focused on examining the local nucleophilicity and electrophilicity (N k and ω k, respectively) for nucleophilic and electrophilic reagents, respectively (Domingo et al., 2021). The computed values of the local electrophilicity for the 2H-2-ethylidene-3-methyl-3,4-dihydronaphthalen-1-one 1 and the local nucleophilicity for the nitrile oxide 2 are presented in Figure 3. The analysis indicates that carbon atom C1 in derivative 1 exhibits a slightly higher electrophilicity than carbon atom C2, while oxygen atom O5 in derivative 2 shows the highest nucleophilicity. These results indicate that the cycloaddition reaction between reagents 1 and compound 2 may occur via the interaction between the carbon atom C1 in reagent 1 and oxygen atom of reagent 2.

Isosurfaces represent Mulliken atomic spin densities with Pk + and Pk − for compound 1 and 2, respectively. All reported values are given in electron volts (eV).

Molecular orbital diagram showing two structures with labeled atoms and data points. The left structure includes atoms labeled C1 and C2 with corresponding values of 0.2884 and -0.0788. The right structure includes atoms labeled C3, N4, and O5 with values 0.0035 and 0.4308. Blue and green lobes represent electron density around the atoms. Reaction profiles for 1,3-DC reaction

The next phase in this mechanistic investigation into the cycloaddition reaction between 1 and 2 aimed to explore the two possible reaction pathways via a single-step mechanism, resulting in the construction of four plausible products. Figure 4 summarizes the attributed activation energy values found through using chloroform as a solvent. Pathway A has a lower activation energy and greater stability than pathway B, even though the Pk + and Pk − Parr functions values suggest that pathway B is more favorable. These results may be attributed to the instability of 3’a, and 3’b products due to steric repulsions between the phenol groups (Figure 4). The results also suggest that TS2 has an activation energy of 14.48 kcal/mol, indicating slightly higher stability, with a difference of 3.2 kcal/mol compared to TS1. These transition states facilitate the formation of product 3a in a more advantageous manner than 3b.

Activation energy diagram of 1,3-DC reaction between reagents 1 and 2, considering chloroform as the reaction medium. All energy values are reported in kcal/mol.

Energy profile diagram illustrating reaction pathways with transition states TS1, TS2, TS3, TS4, and final products 3'a, 3'b, 3a, 3b. The vertical axis shows energy change (Delta Eₐ) in kilocalories per mole. Molecular models of transition states are displayed on the right. Antimicrobial screening Well-diffusion assay

The antimicrobial activity of the synthesized spiro-isoxazoline derivatives was evaluated using the well diffusion method against Gram-positive and Gram-negative bacteria, as well as a yeast strain (Table 2). Among the tested compounds, 3b, bearing a para-chloro substituent (p-ClC6H4), displayed the significant and broadest antimicrobial profile, with IZDs ranging from 8.33 ± 0.57 to 14.00 ± 2.00 mm. In comparison, the standard antibiotics ampicillin and amphotericin B produced significantly larger IZDs (26.66 ± 1.52 to 40.33 ± 0.57 mm). Compound 3c, featuring a para-methoxy substituent (p-OCH3C6H4), showed selective antibacterial activity against S. aureus and B. subtilis, with IZDs ranging from 8.33 ± 1.15 to 9.33 ± 1.52 mm, while remaining inactive against the other microorganisms tested. These findings are consistent with established structure–activity trends, wherein electron-withdrawing groups such as chlorine enhance antibacterial potency by increasing lipophilicity and improving membrane permeation, whereas electron-donating substituents like methoxy typically confer reduced but sometimes more selective activity. Similar observations have been reported for chlorinated and methoxy-bearing isoxazolines and related heterocycles exhibiting antimicrobial properties (Raju et al., 2024).

IZDs (mm) of the tested compounds.

Compounds R Antibacterial activity Antifungal activity
E. coli P. brasiliensis S. aureus B. subtilis C. albicans
3a p-C 6 H 5 --- --- --- --- ---
3b p-ClC 6 H 4 14.00 ± 02.00 a 12.33 ± 0.57 12.66 ± 00.57 14.00 ± 01.00 08.33 ± 00.57
3c p-OCH 3 C 6 H 4 --- --- 08.33 ± 1.15 09.33 ± 01.52 ---
3d p-CH 3 C 6 H 4 --- --- --- --- ---
Ampicillin --- 23.00 ± 01.00 30.00 ± 01.00 27.66 ± 02.08 40.33 ± 00.57 ---
Amphotericin B --- --- --- --- --- 26.66 ± 01.52

Values are expressed as mean ± SD.

 MIC assay

MIC values were determined for the two active compounds 3b and 3c. Compound 3b exhibited remarkable inhibitory activity, with an MIC recorded at 10 μg/mL against Escherichia coli, comparable to that of ampicillin (10 μg/mL). Compound 3b also showed a significant antibacterial effect against Pectobacterium brasiliensis and B. subtilis with MIC values of 10 μg/mL, compared to the positive control, with displayed MIC values of 5 μg/mL, and 2 μg/mL, respectively. Additionally, it showed moderate inhibitory action against S. aureus (90 μg/mL), and Candida albicans (300 μg/mL). Furthermore, 3c displayed antibacterial effectiveness, with MIC values of 50 μg/mL against B. subtilis and 500 μg/mL against S. aureus (Table 3).

MIC (µg/mL) for compounds 3b and 3c.

Compd Antibacterial activity Antifungal activity
E. coli P. brasiliensis S. aureus B. subtilis C. albicans
3b 10 10 90 10 300
3c --- --- 500 50 ---
Ampicillin 10 5 3 2 ---
Amphotericin B --- --- --- --- 2
Molecular interaction analyses

A molecular docking study for the synthesized compounds 3b and 3c, as well as for ampicillin was undertaken to explain its antibacterial activity against two Gram-positive strains, namely, Staphylococcus aureus and Bacillus subtilis. To better understand interactions between the two new molecules synthesized (3b, 3c, and ampicillin) and the targeted proteins, molecular docking was performed to clarify how the chosen ligand interacts with its protein (Figures 5, 6; Table 4).

Interactions between the synthesized compound 3b and 3c, ampicillin and the 1OF0 receptor.

Molecular interaction diagrams and surface representations for Ampicillin-10F0, 3b-10F0, and 3c-10F0. Each figure shows structural interactions like hydrogen bonds and alkyl interactions with color codes for different interactions. The right side features the corresponding surface model with highlighted active sites, showing the spatial conformation and interaction regions with donor and acceptor labels in a gradient from pink to green.

Interactions 2D between the synthesized compound 3b and 3c, ampicillin and the 3VSL receptor.

Molecular docking interactions with 3VSL are shown in three panels. Each panel displays a 2D interaction diagram and 3D surface rendering. Top: Ampicillin binds with hydrogen bonds and carbon hydrogen bonds. Middle: Compound 3b forms hydrogen, alkyl, and pi-alkyl interactions. Bottom: Compound 3c exhibits hydrogen, alkyl, and pi-alkyl bonds. Key depicts bond types and colors.

Molecular docking bending energy results for compounds 3b, 3c, and ampicillin against the two selected proteins.

Ligands Complexes Bending energy
Ampicillin Ampicillin-1OF0 −7.80
3b 3b-1OF0 −8.23
3c 3c-1OF0 −6.62
Ampicillin Ampicillin-3VSL −7.37
3b 3b-3VSL −9.33
3c 3c-3VSL −8.19

The binding energy of the synthesized molecules and the drug ampicillin showed that the two new compounds (3b and 3c) and the drug ampicillin have a good binding affinity (−6.62 and −9.33) with the two targeted proteins. Figure 5 shows the various molecular docking results with receptor 1OF0.

Molecular docking results for the ampicillin-1OF0 complex (Figure 5) reveal two hydrogen bonds with residues Gly-223 and 321, at distances of 2.46 Å and 2.15 Å, respectively. In addition, a pi-lone pair interaction is present with residue Thr-226 at a distance of 2.99 Å. Three π-alkyl interactions are also present with residues His-419 and 497, and residue Ile-494, as well as a carbon-hydrogen bond with residue Cys-322.

Molecular docking results for the 3b-1OF0 complex (Figure 5) reveal a single hydrogen bond and single pi-cation with residues Thr-418 and His-497, at a distance of 2.02 Å and 3.78 Å respectively. In addition, a pi-sulfur interaction is observed with residue Cys-322, at a distance of 5.89 Å. Three π-alkyl interactions are also present with residues Ile-494, Ala-227 and 320. Similarly, molecular docking results for the 3c-1OF0 complex (Figure 5) reveal a single hydrogen bond with residue Thr-377, at a distance of 2.27 Å. In addition, a carbon hydrogen bond is observed with residue Gly-382, at a distance of 3.23 Å. Three π-alkyl interactions are also identified with residues Pro-384, Leu-386 and Ala-375. We observed that the two molecules synthesized, 3b and 3c, interact with the same residues as ampicillin, namely, threonine, cysteine and alanine. This confirms that these two compounds are indeed localized at the active site of the target protein. So, these interactions may contribute to the inhibition of the targets against Bacillus subtilis.

Molecular docking results for the three molecules studied show that all the selected molecules showed outstanding docking scores (Table 4) against antibacterial activity targeting Staphylococcus aureus (Figure 6).

Molecular docking results for the ampicillin-3VSL complex (Figure 6) reveal three hydrogen bonds with residues Pro-660, Thr-621, and Lyr-618, at distances of 2.08 Å, 2.20 Å, and 2.22 Å, respectively. Molecular docking results for the 3b-3VSL complex (Figure 6) reveal three hydrogen bonds with residues Tyr-605, Val-606, and Thr-603, at distances of 2.60 Å, 2.24 Å, and 2.02 Å, respectively. In addition, two π-alkyl interactions are also present with residues Val-658 and Pro-660. Similarly, molecular docking results for the 3c-3VSL complex (Figure 6) reveal two hydrogen bonds with residues Thr-619, and Thr-621, at distances of 1.75 Å, and 2.92 Å, respectively. In addition, a free π-electron interaction (π-lone pair) is observed with residue Thr-621, at a distance of 2.92 Å. Two π-alkyl interactions are also identified with residues Pro-606 and Val-606. We noted that the two molecules synthesized, 3b and 3c, interact with the same residues as ampicillin, namely, threonine and proline. This confirms that these two compounds are indeed localized at the active site of the target protein. Molecular docking studies against both bacterial strains revealed that the two synthesized compounds possess antibacterial potential and interact with amino acid residues like those targeted by the reference antibiotic, ampicillin.

 POM analysis of compounds

The POM Theory was developed by our group, in collaboration with NCI and TAACF of the United States of America. The principal goal is to demonstrate differences between various classes of commercial drugs, based on their physico-chemical properties and atomic charges of each pharmacophore site (Figure 7).

Organigram of POM Theory showing the geometry and atomic charge of pharmacophore site of antibacterial (Grib et al., 2020; Lakhrissi et al., 2022; Mabkhot et al., 2014; Rbaa et al., 2019), antifungal (Al-Maqtari et al., 2017; Rachedi et al., 2020), antiviral and antiparasitic (Aljofan et al., 2014; Jarrahpour et al., 2019; Kawsar et al., 2022; Khodair et al., 2021) and antitumor agents (Al-Maqtari et al., 2017; Aljofan et al., 2014; Bechlem et al., 2020; Bennani et al., 2013; Grib et al., 2020; Jarrahpour et al., 2019; Kawsar et al., 2022; Khodair et al., 2021; Lakhrissi et al., 2022; Mabkhot et al., 2014; Rachedi et al., 2020; Rbaa et al., 2019).

Diagram showing the POM theory applied to pharmacophore sites for antitumor, antibacterial, antifungal, antiviral, and anti-HIV agents. Four circular structures (I-IV) are depicted, each associated with different agents: antitumor (magenta), antibacterial (red), antifungal/antiviral (black), and anti-HIV (blue). Each structure shows electronegative and electropositive regions with labeled distances between specific atoms, indicating their roles in interacting with pharmacophore sites. The POM theory is central, linking these structures with arrows. Osiris calculations of toxicity and drug-score of compounds

When a structure is valid, the OSIRIS Property Explorer allows chemical structures to be determined and instantly calculates a variety of drug-relevant properties. The outcomes of predictions are colored-coded and rated. Red indicates properties that have a significant risk of undesirable outcomes, such as mutagenicity or poor intestinal absorption. Alternatively, drug-conformant behavior is indicated by a green hue (Table 5). None of the compounds of series 3a-d have side effect and their drug score is encouraging (40%<DS<51%) but their bioavailability is not optimal (cLogP >5).

Osiris calculations of toxicity and Drug-score of compounds 3a-d.

Toxicity risks assessment shows all green indicators for mutagenic, tumorigenic, irritant, and reproductive effects. Chemical properties include cLogP at 5.06, solubility at −5.58, molecular weight at 319.0, topological polar surface area (TPSA) at 38.86, drug-likeness at 0.73, and drug-score at 0.4. Toxicity risks are indicated as mutagenic, tumorigenic, irritant, and reproductive, with green circles suggesting low risk. Metrics include cLogP at 5.32, solubility at negative 6.97, molecular weight at 339.0, TPSA at 38.66, druglikeness at 3.33, and drug-score at 0.42, each represented by color-coded bars.
Compound 3a Compound 3b
Chart displaying toxicity risks and molecular properties of a compound. Toxicity risks show green indicators for mutagenic, tumorigenic, irritant, and reproductive effective. Measurements include cLogP at 4.66, Solubility at −5.25, Molecular weight at 335.0, TPSA at 47.89, Druglikeness at 2.27, and Drug-Score at 0.51. Toxicity risk assessment and molecular property analysis are shown. No risks for mutagenic, tumorigenic, irritant, or reproductive effects. Values: cLogP 5.06, solubility −5.58, molweight 319.0, TPSA 38.66, drug-likeness 0.73, drug-score 0.4.
Compound 3c Compound 3d
Molinspiration calculations of molecular properties of compounds

To control of bioavailability of candidate drugs, it is of importance to calculate all parameters of Lipinski 5 rules, via Molinspiration program (Table 6). The consultation of Table 6 shows that all compounds meet the criteria of the bioavailability (NV < 2).

Molinspiration calculations of physico-chemical properties of compounds 3a-d according to Lipinski 5 rules.

Compd 3D structure Lipinski 5 rules calculations
3a Space-filling molecular model displaying a carbon-heavy structure. Gray spheres represent carbon atoms, white spheres denote hydrogen atoms, one blue sphere indicates a nitrogen atom, and two red spheres signify oxygen atoms. Molecular model featuring a complex organic structure with a hexagonal benzene ring and a series of interconnected rings and chains. Atoms are represented by grey (carbon), red (oxygen), blue (nitrogen), and white (hydrogen) spheres. miLogP 4.35 TPSA 38.67 natoms     23 MW 305.38 nON        3 nOHNH             0 nviolations          0 nrotb             1 volume 285.08
3b Molecular model showing colored spheres representing atoms of a chemical compound. Gray spheres are carbon atoms, white are hydrogen, red is oxygen, blue is nitrogen, and magenta represents another atom type. Molecular structure model showing a complex molecule with carbon atoms in gray, hydrogen in light gray, nitrogen in blue, oxygen in red, and a halogen atom in pink, forming a three-dimensional arrangement. miLogP 5.03 TPSA 38.67 natoms     24 MW 339.82 nON        3 nOHNH             0 nviolations          1 nrotb             1 volume 298.62
3c Ball-and-stick model of a molecular structure with spheres representing different atoms. Gray spheres are carbon, red are oxygen, blue is nitrogen, and white are hydrogen. Molecular model depicting a complex structure with gray sticks representing carbon atoms, red for oxygen, blue for nitrogen, and white for hydrogen. The structure has a network of interconnected rings with branching extensions. miLogP 4.41 TPSA 47.90 natoms     25 MW 335.40 nON        4 nOHNH             0 nviolations          0 nrotb             2 volume 310.63
3d Molecular model of a compound showing gray, white, red, and blue spheres. The gray and white spheres represent the main structure, while red and blue spheres highlight specific atoms, likely oxygen and nitrogen. Molecular structure model featuring carbon, hydrogen, nitrogen, and oxygen atoms. Carbon atoms are gray, hydrogen is white, nitrogen is blue, and oxygen is red. The structure shows a complex organic compound formation. miLogP 4.80 TPSA 38.67 natoms     24 MW 319.40 nON        3 nOHNH             0 nviolations          0 nrotb             1 volume 301.64
Atomic charge calculations and pharmacophore site identification

The identification of the pharmacophore site for each molecule was based on the X and Y atomic charges of each pocket and the corresponding (X-Y) distance. There is a coexistence of two combined antifungal (O1δ−---O2δ−) and antiviral (O1δ−---N1δ−) pharmacophore sites, which results in a major issue due to the two-methyl substituents on the central rings (Table 7). For this reason, the potential of this series is likely better as an antiviral than antibacterial and antifungal agents; which requires further experimental validation.

Atomic charge and pharmacophore sites identification of compounds 3a-d.

Compd Atomic charge Pharmacophore sites
3a Molecular structure diagram with connected spheres representing atoms. Lines indicate bonds with associated numerical charges in colored boxes. Red and blue spheres signify specific atoms, such as oxygen and nitrogen, with their respective charges. Molecular structure model with gray spheres representing atoms connected by bonds. A translucent surface highlights regions with different colors, such as red and blue, indicating potential chemical activity. A green oval overlays part of the structure.
3b Molecular structure diagram featuring atoms connected by bonds. Carbon atoms are gray, oxygen atoms are red, a nitrogen atom is blue, and a chlorine atom is green. Numerical values indicate partial charges near each atom. Chemical structure diagram showing a molecular model with a color gradient background, highlighting atoms in different colors. A green area overlays part of the molecule, possibly indicating an area of interest or interaction.
3c Molecular structure diagram displaying atoms as spheres connected by lines representing bonds. Numerical values near atoms indicate charges or properties. Red, blue, and gray spheres depict different elements, with red lines connecting some pairs, indicating significant interactions. Molecular structure model featuring a 3D representation of atoms connected in a chain. A green oval highlights a specific area on the structure, with gray, red, and blue atoms indicating different elements. Surrounding shading suggests an electron cloud.
3d Molecular structure diagram with gray, red, and blue spheres representing atoms in a compound. Numerical values near bonds indicate charge distributions, with some values in red and others in blue. Molecular structure diagram with atoms represented as spheres connected by lines. A green oval highlights a specific area on the structure. Surrounding areas are shaded in blue and red tones.
3a-d Chemical structure diagram illustrating pharmacophore sites. Red highlights an antifungal site with O1 and O2, while blue highlights an antiviral site with O1 and N1. Both are based on POM Theory. Common pharmacophore sites of compound 3a-d
Conclusion

A new series of spiroisoxazoline derivatives was synthesized through a regio- and diastereoselective 1,3-DC reaction of the arylnitriloxides as dipoles and (E)-2-ethylidene-3-methyl-3,4-dihydronaphthalene-1(2H)-one as a dipolarophile. The structures and the observed regiochemistry of the synthesized spiroisoxazolines were established using standard spectroscopic methods, and further validated by elemental analysis (EA), and HRMS. Furthermore, mechanistic studies were carried out using DFT calculations with the B3LYP/6-31G (d,p) to gain deeper insight into the regioselective synthesis of new spiro-compounds. The theoretical findings obtained align closely with the experimental observations. The in vitro antibacterial screening of the synthesized compounds against a range of bacterial strains, employing agar-well diffusion and microdilution methodologies, revealed that the spiroisoxazoline 3b demonstrated antimicrobial action against all the pathogenic strains tested, while compound 3c exhibited antibacterial activity solely against the two Gram-positive bacteria tested. In silico studies were also carried out to rationalize the experimental findings and provide mechanistic insight. POM analyses of the relative antimicrobial activity of these derivatives were also performed. Interestingly, drug-likeness analysis suggested that the tested spiro-isoxazolines would require structural optimization to yield derivatives with improved oral bioavailability and favorable brain penetration properties. Therefore, the results of the present investigation suggest that the studied congeners represent promising antiviral candidates, warranting further in-depth investigation. The current study provides important insights into the origins of the modest antimicrobial potential of spiro-compounds, thereby supporting their use as scaffolds in the rational design and development of more potent antiviral drug candidates. Taken together, the combined experimental and theoretical results provide a foundation for the rational design and development of new antiviral spiro-compounds with promising therapeutic potential.

 Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

 Author contributions

RB: Conceptualization, Investigation, Resources, Writing – original draft. SA: Investigation, Resources, Writing – original draft. MR-R: Formal Analysis, Software, Writing – original draft. NA: Data curation, Software, Writing – original draft. LB: Writing – original draft, Data curation, Formal Analysis. MC: Investigation, Validation, Writing – review and editing. ME: Validation, Visualization, Writing – review and editing. ML: Resources, Validation, Writing – review and editing. TB: Investigation, Validation, Writing – original draft. DB: Validation, Visualization, Writing – original draft. AA: Funding acquisition, Validation, Writing – original draft. MA-S: Funding acquisition, Investigation, Writing – review and editing. JG: Formal Analysis, Funding acquisition, Writing – review and editing. GA: Conceptualization, Project administration, Supervision, Writing – review and editing.

 Acknowledgements

The authors would like to thank the University of Dhar El Mehraz, Sidi Mohammed Ben Abdellah, and the Euro-Mediterranean University of Fez (Morocco) for funding this research as part of the Fundamental Research Facilities program. The authors gratefully acknowledge the staff members of the “Cité de l'Innovation” of Sidi Mohamed Ben Abdellah University (Morocco). The authors are also grateful to TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources) for providing the facilities for the DFT calculations reported in this work.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Generative AI statement

The authors declare that no Generative AI was used in the creation of this manuscript.

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 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2025.1740409/full#supplementary-material

Edited by: Jian-Wei Han, East China University of Science and Technology, China

Reviewed by: Nosrat Madadi Mahani, Payame Noor University, Iran

Aram Rahman, Maulana Azad National Urdu University, India

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