Mechanistic Insights into (3+2) Cycloaddition of Glutaraldehyde-N-Aryl Nitrone with Cinnamaldehyde: Electron Density, Docking, and Molecular Dynamics Analysis

Mechanistic Insights into (3+2) Cycloaddition of Glutaraldehyde-N-Aryl Nitrone with Cinnamaldehyde: Electron Density, Docking, and Molecular Dynamics Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanistic Insights into (3+2) Cycloaddition of Glutaraldehyde-N-Aryl Nitrone with Cinnamaldehyde: Electron Density, Docking, and Molecular Dynamics Analysis Raad Nasrullah Salih, Haydar Mohammad-Salim, Muheb Algso This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7773346/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Structural Chemistry → Version 1 posted 9 You are reading this latest preprint version Abstract This research provides a thorough computational analysis of the [3 + 2] cycloaddition (32CA) reaction involving Glutaraldehyde-N-aryl nitrone ( GN-1 ) with Cinnamaldehyde ( CA-2 ), utilizing Molecular Electron Density Theory (MEDT) as the interpretive framework. However, the potential energy surface analysis identified four stereoisomeric pathways, with the ortho - endo channel becoming thermodynamically and kinetically better. However, Global Electron Density Transfer (GEDT) values at the transition states (TSs) showed a clear forward electron density flux (FEDF), confirming the polar nature of the main mechanism. Electron Localization Function (ELF) with Bonding Evolution Theory (BET) analyses highlighted asynchronous bond formation along the preferred pathway. The stability of the cycloadducts was further assessed through molecular docking against the EGFR L858R mutant (PDB ID: 2ITZ), where compound IC-6 exhibited the strongest binding affinity. Molecular dynamics (MD) simulations of the IC-6 /EGFR complex over 100 ns validated its stable interaction profile and dynamic conformational behavior within the binding site. Furthermore, Absorption, Distribution, Metabolism, Excretion, with Toxicity (ADMET) predictions confirmed favorable drug-likeness with pharmacokinetic characteristics for synthesized compounds. Overall, integration of GEDT, FEDF, and MD provides deep mechanistic insights into the reaction pathway, while highlighting the therapeutic potential of the resulting isoxazolidine derivatives. GEDT FEDF Molecular Docking ADMET Molecular Dynamics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction A three-atom component (TAC) reacts with alkene via 32CA, considered one of the most effective ways to synthesize five-membered heterocycle compounds. Additionally, this reaction can produce cyclic compounds via great regio- and stereoselectivity in organic chemistry synthesis. This reaction can form cyclic compounds with high regio- and stereoselectivity [ 1 ]. Nitrones serve as important TACs commonly used in 32CA reactions. These compounds produce pharmacologically active isoxazolidines and isoxazolines, distinguished by their stereochemistry regioselectivity [ 2 ]. In the last ten years, detailed studies using conceptual density functional theory (DFT) continuously nitrone cycloadditions had continuously revealed a strong relationship between experimental outcomes with DFT-predicted selectivity [ 2 , 3 ]. However, research released post-2003 categorizes TACs and alkenes based on absolute electrophilicity, showing electron density changes during cycloaddition [ 4 ]. However, since 2003, numerous pathways for 32CA responses have been explored and suggested. Subsequently, in 2014 and 2017, researchers hypothesized one-step with two-step polar mechanisms for 32CA reactions involving nitrones [ 5 , 6 ]. Subsequently, Domingo illustrated the MEDT in 2016, emphasizing how electron density fluctuations influence organic reaction reactivity [ 7 ]. Particularly, Domingo’s MEDT [ 8 – 10 ] is a crucial framework for grasping reactivity in molecular organic chemistry. Consequently, it demonstrates how fluctuations in electron density drive reactivity. Moreover, MEDT research has elucidated the mechanisms of 32CA reactions, demonstrating that they occur through the sequential alteration of bonds throughout one-step reactions. Additionally, distinguishes them starting the coordinated pericyclic mechanism described by Woodward with Hoffman 1969. Although certain 32CA reactions are non-polar, Parr functions help clarify the regioselectivity seen in polar conditions. Additionally, in 2014, Tang and colleagues conducted a DFT study to examine the reaction between 1,3-dialkynes also ammonia derivatives [ 11 , 12 ]. Normally, Jasinski (2018) proposed that in the polar nitrone 32CA reactions via arylacetylenes, both one-step and two-step reactions occur simultaneously, competing with each other [ 13 ]. Moreover, new investigations in 2017 and 2018 has shown nitrone 32CA reactions are one-step, two-stage with asynchronous bond development [ 11 , 14 , 15 ]. Generally, a wide range of chemicals were used to come up with a standard way to group TACs into four groups: [ 7 , 15 , 16 ] pseudodiradical ( pdr ) type [ 17 ], pseudo ( mono ) radical ( pmr ) type, carbenoid ( cb ) type also zwitterionic ( zw ) type [ 15 ]. In addition, relative reactivity movement goes in the order: pdr type > pmr type ≈ cb type > zw type. Therefore, MEDT involves understanding reactivity, indicating that shifts in electron density cause molecular changes in reactivity. In addition, A regular of quantum chemical tools, including ELF [ 18 , 19 ], However, ELF, global and local reactivity indices [ 7 , 20 ], and Laplacian of electron density [ 21 , 22 ]. In this contribution, the MEDT is employed to analyze the 32CA between Glutaraldehyde-N-aryl nitrone GN-1 and Cinnamaldehyde CA-2 (Scheme 1 ). Moreover, the 32CA reaction remained recent established by Sivadharani and Jayapradha [ 23 ] as a novel methodology for producing isoxazolidine, which involves adding nitrone GN-1 and CA-2 in catalyst-free conditions (Scheme 1 ). A mixture of nitrone GN-1 and CA-2 is refluxed in toluene (50ml) for the time period specified in Scheme 1 . After 18 hours, the reaction afforded the major product, (3S,4R,5S)-4-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-5-carbaldehyde ( IC-3 ), in 89% yield. There were also small amounts of another product seen to form [ 23 ]. This work objects to demonstrate the energetics also reaction mechanism of the computationally 32CA between nitrone GN-1 and CA-2 . Furthermore, the interactions between molecules (3S,4R,5S)-4-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-5-carbaldehyde ( IC-3) , (3R,4S,5R)-4-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-5-carbaldehyde ( IC-4) , (3S,4R,5S)-5-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-4-carbaldehyde ( IC-5) and (3R,4S,5R)-5-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-4-carbaldehyde ( IC-6) through the 2ITZ structures were estimated via molecular docking simulations. However, EGFR L858R Mutation with Gefitinib Protein Data Bank (PDB ID: 2ITZ ): This entry showcases the EGFR kinase domain harboring the L858R mutation, a common alteration in lung cancer, in complex with the drug gefitinib (Iressa). Such mutations can affect drug binding and influence treatment efficacy [ 24 ]. Moreover, the prediction of ADMET properties are crucial in drug investigation and progress. Consequently, once docking, the ADMET of IC-3 , IC-4 , IC-5 , also IC-6 were assessed and compared with control molecules for each target. Moreover, this evaluation provided a significant understanding of their capabilities for other advancement in medication detection and progress. Altogether, manuscript integrates mechanistic organic chemistry with MD–based validation to highlight both theoretical insights and therapeutic potential of the synthesized isoxazolidine derivatives. 2. Computational method Improved chemical structures were obtained utilizing M06-2X-D3/6-311G++(d, p) [ 25 ] theoretical level, along with Berny analytical gradient optimization [ 26 , 27 ]. However, because they didn't have any imaginary frequencies. Moreover, presence of one imaginary frequency identified TSs. However, to account dispersion effects influencing the stability of complexes and intermediates, the D3 correction was useful [ 28 ]. In addition, Geometry optimizations used default convergence criteria for geometry, integration grid, as well as SCF procedures in Gaussian 16. Therefore, open-shell systems which handled utilizing a spin-restricted formalism. However, Intrinsic Reaction Coordinate (IRC) analyzed the whole reaction path. Therefore, this approach enables the identification of the reaction path also intermediate states, beside their associated activation energies. All computational analyses were conducted utilizing Gaussian 16, Revision B.01 [ 29 – 33 ]. Moreover, the entire reaction path examined utilizing the IRC methodology. However, this method enables a clearer view of the reaction path and assists in identification of intermediate states, along with their corresponding activation energies [ 34 ]. To characterize the polarity of the reaction, GEDT [ 35 ], the TS was found by adding up the natural atomic charges (q), which were found in a natural bond orbital (NBO) [ 36 ] study of the atoms within a separately framework (f); namely, i.e., \(\:\text{G}\text{E}\text{D}\text{T}=\sum\:_{i\in\:f}{q}_{i}.\:\:\) Furthermore, Positive values show electronic flux from the framework to the alternative, conversely. Identically, utilizing the framework of conceptual DFT, various descriptors of electronic structure and reactivity were assessed, incorporating the energies of the highest occupied molecular orbital (HOMO, εH) with lowest unoccupied molecular orbital (LUMO, εL). Conversely, this includes LUMO-HOMO gap (gap = εL - εH), global electrophilicity (ω = µ2/2η) [ 37 ], and chemical potential [µ = (εH + εL)/2] [ 38 ] with the global hardness (η = ε L - ε H ) [ 39 ]. Chemical softness (S) [ 40 ] is the opposite of chemical hardness and indicates susceptibility of a molecule's electron density to distortion or perturbation. However, the nucleophilicity index (N) was determined using the formula N = ε H (Nu) − ε H (TCE), where Nu represents nucleophile while TCE denotes tetracyanoethylene reference [ 5 ]. Nonetheless, neutral species and their radical anions and cations were used in natural population analysis (NPA) to calculate Parr, Pearson, and Yang local indices [ 36 , 41 ]. Moreover, atomic spin density (ASD) characterizes these functions to assess polarity reactions. While Parr functions, well-defined as \(\:{P}_{k}^{+}\) for electrophilic attack also \(\:{P}_{k}^{-}\) for nucleophilic attack, were expressed relative to the respective population \(\:{q}_{k}\:\) (N) of k th atomic site of the N-electron system, \(\:{P}_{k}^{+}={q}_{k}(N+1)-{q}_{k}\left(N\right)\) and \(\:{P}_{k}^{-}={q}_{k}\left(N\right)-{q}_{k}(N-1)\) respectively. Similarly, the local electrophilicity, \(\:{\omega\:}_{k}\) , which gained via \(\:{\omega\:}_{k}=\:\omega\:\:\times\:\:{P}_{k}^{+}\) , also the local nucleophilicity indices, \(\:{N}_{k}\) , which determined via \(\:{N}_{k}=N\times\:{P}_{k}^{-}\) [ 5 , 42 ] for a specific input reaction, Domingo et al. [ 43 ] suggested a local reactivity difference index \(\:{R}_{k}\) , illustrated as \(\:{R}_{k}={(\omega\:}_{k}+{N}_{k})/2\) , utilized to identify the species' greatest electrophilic and nucleophilic centers. However, all computations used Gaussian16. Moreover, Non-Covalent Interaction (NCI) analysis involved performance on each optimized TS geometry and using a single point calculation. Furthermore, the related graphs of electron density [ρ(r)] against the reduced density gradient (RDG) \(\:s\left(r\right)=\left|{\nabla\:}_{\rho\:}\left(r\right)/2{\left(3{\pi\:}^{2}\right)}^{1/3}{\rho\:\left(r\right)}^{4/3}\right|\:\) which generated utilizing the NCIPLOT software [ 44 ]. Additionally, the ELF of the molecule was determined with Topmod [ 18 , 45 ]. Above all, the Multiwfn program [ 18 , 19 , 46 ] was employed for the topological study of the ELF besides the Quantum Theory of Atoms in Molecules (QTAIM). In addition, natural population analysis (NPA) [ 36 , 41 ] was done at the TSs to find out what the atoms' natural charges ( q ) are. A thorough examination of the BET [ 47 ] was done to investigate the bonding transformations throughout experimentally established reaction path, including nitrone GN-1 and CA-2 . Moreover, the ELF domains were pictured via VMD [ 48 ]. Graphical illustrations of the ELF attractor basin placements were created utilizing the GaussView6 program [ 49 ]. In addition, visualization of ELF domains was accomplished with Paraview [ 50 ]. Following optimization, molecules IC-3 , IC-4 , IC-5 and IC-6 were employed for molecular docking studies, which were achieved via Cresset Flare V9 [ 51 ]. Moreover, three-dimensional (3D) structures of the molecular targets were acquired from Protein Data Bank (PDB) ( www.rcsb.org ). Additionally, the schematic depiction of the chemical pathways was produced utilizing PerkinElmer ChemDraw. Moreover, forecasts of ADMET characteristics (absorption, distribution, metabolism, excretion, with toxicity) performed for combined drugs utilizing the SwissADME platform also the ADMETlab 3.0 web server. However, A PASS (Prediction of Activity Spectra for Substances) study was conducted to forecast their biological activities. Furthermore, they were subsequently subjected to MD simulations using Gromacs 2025 on a GPU-accelerated system [ 52 ]. 3. Results and discussion This paper consists of eleven main sections number: (1) The ELF [ 18 ] is looked at to learn more about the electronic structure of nitrone GN-1 and CA-2 when they are in their ground state (GS). (2) Furthermore, CDFT [ 38 , 53 ] has been used to calculate the polar attributes and electronic distribution of the reactions. (3) Researchers have thoroughly studied the potential energy surface (PES) to discovery stationary sites along the possible reaction paths of the 32CA reactions in the investigation. (4) Moreover, the electronic structure of the identified TSs was examined utilizing ELF topological analysis [ 18 ] and QTAIM [ 46 ] parameters. (5) A BET study was conducted along the reaction path. (6) The study of NCI is very significant for understanding how the stability and behavior of molecular systems are affected. (7) An elementary examination of the Atoms-in-Molecules (AIM) at the TSs was conducted. (8) Molecular docking studies examined interactions between cycloadducts IC-3 , IC-4 , IC-5 , with IC-6 (2ITZ). (9) Additionally, the material's drug-like properties were evaluated, along with its ADMET assessment characteristics. (10) PASS analysis is conducted using the PASS online platform. (11) Finally, Molecular dynamics (MD) allows the exploration of stability and dynamics of complex systems. It provides insights into structural stability, flexibility, and interactions of biomolecules under near-physiological conditions. 3.1 A study of the ELF topology of nitrone GN-1 with Cinnamaldehyde CA-2 Becke and Edgecombe [ 18 ] originally formulated the ELF, which Silvi and Savin later enhanced [ 19 ]. While ELF has been established as a precise computational tool for studying the electronic structure of chemical systems processes. However, ELF efficiently delineates electronic regions like core, bonding, with non-bonding areas as attractors. ELF delineates basins of attractors as areas where the likelihood of locating electron pair is maximized. Otherwise, these basins are divided into core basins (C(...)) also valence basins (V(...)). Moreover, valence basins were categorized through their synaptic order, indicating the number of atomic valence shells involved. Nevertheless, it can be monosynaptic, disynaptic, trisynaptic, etc [ 19 ]. Domingo's examination of ELF topology provides key insights for classifying 32CA reaction TACs as pseudo-diradical , pseudo(mono)radical , carbenoid , with zwitterionic [ 15 , 54 , 55 ]. Additionally, monosynaptic basins, indicated V(A), represent lone pairs or non-bonding regions. Moreover, disynaptic basins, marked V(A,B), associate the centers of two nuclei, A with B, while showing the bonding area between them. A monosynaptic basin with fewer than one electron is linked to a pseudoradical center [ 17 , 56 ], while one with more than one electron is linked to a carbenoid center. Moreover, this study analyzes electronic structures of nitrone GN-1 with CA-2 involved 32CA reactions using ELF topological analysis. In addition, ELF topology of nitrone GN-1 demonstrations two monosynaptic basins, V(O1) also V′(O1), with 5.92 e, and two disynaptic basins, V(N2,C3), also V(N2,O1), with 3.77 e and 1.38 e. e, respectively. Furthermore, monosynaptic basins V(O1) also V′(O1) denote the nonbonding electron density associated with the O1 oxygen atom. Similar to this, underpopulated N2-C3 double bond is linked to disynaptic basin V(N2,C3), whereas underpopulated N2–O1 single bond is linked to V(N2,O1). Consequently, nitrone GN-1 can be able to categorized as a zwitterionic TAC because of deficiency of pseudoradical or carbenoid centers [ 10 , 57 ]. Moreover, ELF of CA-2 displays a V(C4,C5) disynaptic basin that integrates 3.30 electrons and is associated to the ethylene C4 and C5 atoms' double bonding area. In addition, the ELF valence basin populations represent the electron localisation in different molecular regions, helping to understand bonding characteristics and electronic structure. Consequently, Fig. 1 demonstrations Lewis-like structures of nitrone GN-1 and CA-2 reactants, with atomic charges from NBO analysis. In GN-1 , oxygen (O1) has a charge of − 0.61 e, nitrogen (N2) + 0.059 e, carbon (C3) + 0.073 e. In CA-2 , carbon atoms take charges of − 0.11 e and − 0.32 [ 58 – 60 ]. 3.2 Analysis of CDFT Indices for Nitrone GN-1 with Cinnamaldehyde CA-2 An examination of the CDFT [ 38 , 53 ] indices offers a preliminary insight into the reactivity of organic compounds in polar processes. Admittedly, concept of "Conceptual DFT," introduced by Parr, has been widely utilized to evaluate the chemical reactivity of structures included in 32CA reactions [ 7 , 20 , 61 , 62 ]. For the CDFT investigation, Domingo's M06-2X-D3/6-311 + + G(d,p) standard scales for electrophilicity and nucleophilicity indices were employed [ 63 – 65 ]. Using M06-2X-D3/6-311 + + G(d,p) level of theory, CDFT indices for nitrone GN-1 with CA-2 calculated based on established reactivity scales. [ 53 , 63 , 66 , 67 ]. Similarly, the chemical potential (µ), chemical hardness (η), chemical softness (S), electrophilicity (ω), also nucleophilicity (N) expressed in electron volts (eV) units. Moreover, Table 1 indicates that chemical hardness (η) for nitrone GN-1 is about 7.11 eV. However, electronic chemical potential (µ) of nitrone GN-1 is − 4.24 eV, upper that of CA-2 . The calculated value for CA-2 is − 4.34 eV. Overall, CA-2 has the highest electronic chemical potential. Additionally, the following is a record of chemical softness (S) values that were determined for the reactants: nitrone GN-1 has a value of 0.14 eV, whereas CA-2 exhibits the highest value at 0.16 eV. On the other hand, nitrone GN-1 takes an electrophilicity (ω) index [ 37 ] of 1.27 eV, categorizing it as a moderate electrophile based on electrophilicity scale [ 53 ]. Additionally, chemical softness (S) values for reactants show nitrone GN-1 at 0.14 eV and CA-2 at 0.16 eV, the highest. Nitrone GN-1's electrophilicity (ω) index is 1.27 eV [ 37 ] making it a moderate electrophile per electrophilicity scale [ 53 ]. Furthermore, nucleophilicity index of this substance is 3.11 eV, which indicates that it is a strong nucleophile according to nucleophilicity scale [ 53 ]. Overall, the electrophilicity (ω) index of CA-2 was 1.53, reflecting its strong electrophilic character. Notably, nitrone GN-1 shows higher nucleophilicity than CA-2 , indicating a greater propensity to function as a nucleophile. The GEDT [ 35 ] will occur from nitrone GN-1 to CA-2 . Consequently, it is expected that the electronic flux goes among nitrone GN-1 to CA-2 , which is in accordance with the findings on electronic chemical potential (µ). Additionally, values of (η) support the nucleophilic nature nitrone GN-1 with the electrophilic nature of CA-2 . However, 32CA reaction demonstrates clearly polar behavior, termed forward electron density flux (FEDF), resulting from nitrone GN-1 's strong nucleophilicity and the significant electrophilicity of CA-2 . Table 1 Calculations at M06-2X-D3/6-311 + + G(d,p) level provided ground-state electronic properties of nitrone GN-1 and CA-2 , including chemical hardness η, chemical potential µ, chemical softness S, global electrophilicity ω, with global nucleophilicity N, each shown in eV. Reagent η S µ ω N GN-1 7.11 0.14 -4.24 1.27 3.11 CA-2 6.18 0.16 -4.34 1.53 3.10 Furthermore, when two species with different properties interact in a polar 32CA reaction, the preferred pathway is typically determined by the interaction between the location that is the most electrophilic for one species and the site that is the most nucleophilic for the other species [ 68 ]. Nevertheless, several studies emphasize the significance of electrophilic ( \(\:{P}_{\text{k}}^{+}\) ) with nucleophilic ( \(\:{P}_{\text{k}}^{-}\) ) Parr functions [ 42 ]. Using additional spin electron density from GEDT, these functions are one of the best dependable ways to evaluate local reactivity in polar also ionic systems processes. However, Table 2 shows Parr nucleophilic ( \(\:{P}_{\text{k}}^{-}\) ) also electrophilic ( \(\:{P}_{\text{k}}^{+}\) ) functions, local electrophilic (ωk), nucleophilic (Nk) indices, the local reactivity difference index (R k ). In addition, examination of electrophilic ( \(\:{P}_{\text{k}}^{+}\) ) with nucleophilic ( \(\:{P}_{\text{k}}^{-}\) ) Parr functions in nitrone GN-1 shows that C3 carbon atom is the greatest electrophilic site, exhibiting ( \(\:{P}_{\text{k}}^{+}\) ) = 0.37, whilst the least nucleophilic site is O1, with ( \(\:{P}_{\text{k}}^{-}\) ) = -0.031. In the instance of CA-2 , the C4 is designated as least electrophilic site ( \(\:{P}_{\text{k}}^{+}\) = 0.25), whereas the most nucleophilic site is also C4 ( \(\:{P}_{\text{k}}^{-}\) = 0.038) [ 69 ]. The R k values show the most favorable interactions between electrophiles and nucleophiles along the reaction pathway. It was found that the main interaction in this condensation occurs among the electrophilic center, C5 ( R k = 0.23) of CA-2 , nucleophilic center, O1 ( R k = -0.038) of nitrone GN-1 . Figure 2 illustrates the 3D representation of the nucleophilic ( \(\:{P}_{\text{k}}^{-}\) ) also electrophilic ( \(\:{P}_{\text{k}}^{+}\) ) Parr functions between nitrone GN-1 and CA-2 . Table 2 There are local electrophilic and nucleophilic indices (ωk, Nk) with local reactivity difference indices ( R k ) values for M06-2X-D3/6-311 + + G(d,p). These include nucleophilic ( \(\:{P}_{\text{k}}^{-}\) ) with electrophilic ( \(\:{P}_{\text{k}}^{+}\) ) Parr functions. Reactant Center \(\:{P}_{\text{k}}^{+}\) \(\:{P}_{\text{k}}^{-}\) ω k N k R k GN-1 O1 0.17 -0.32 0.22 -0.99 -0.38 N2 0.24 0.18 0.30 0.55 0.43 C3 0.37 -0.25 0.47 -0.79 -0.15 CA-2 C4 0.15 0.038 0.23 0.12 0.17 C5 0.25 0.03 0.38 0.093 0.23 GN-1 CA-2 Scheme 2 . RMMRs ( \(\:{\text{R}}_{\text{k}}\) molecular maps of reactivity) for the reaction of nitrone GN-1 and CA-2 . \(\:{\text{R}}_{\text{k}}\) >0, indicated in red, identifies electrophilic centers, while \(\:{\text{R}}_{\text{k}}\) < 0, shown in blue, indicates nucleophilic centers. However, Chattaraj introduced local reactivity modification index in 2012 [ 43 ], a tool for predicting the local electrophilic aslo nucleophilic activation of organic compound molecules [ 53 ]. In Scheme 2, nitrone GN-1 shows \(\:{\text{R}}_{\text{k}}\) values of -0.38 for O1 and − 0.15 for C3, indicating a tendency toward nucleophilicity. Conversely, for CA-2 , the \(\:{\text{R}}_{\text{k}}\) values of C4 also C5 are 0.17 and 0.23, respectively, implying that C3 and C4 behave as electrophiles. 3.3 Exploring the Potential Energy Surface (PES) Due to asymmetry of the reactants, the 32CA (1,3-dipolar cycloaddition) reaction among nitrone GN-1 with CA-2 can proceed through four competing reaction paths. These encompass two regioisomeric pathways: ortho and meta , as well as two stereoisomeric orientations: endo and exo . As a result, the molecules ( TS1-ex , TS1-en , TS2-ex , with TS2-en) have been identified, each leading to a corresponding cycloadduct: ( IC-3 , IC-4 , IC-5 , with IC-6 ) respectively. These TSs and products have been identified and described (see Scheme 3 and Table 3 ). However, Table 3 summarizes the energetic also electronic properties of the 32CA stationary points reaction. Nonetheless, these values were computed in toluene using M06-2X-D3/6-311 + + G(d,p) theoretical method. However, the table shows cycloadducts ( IC-3 to IC-6 ), TSs ( TS1-en , TS1-ex , TS2-en , TS2-ex ), the data on electronic energy (ΔE), enthalpy (ΔH), Gibbs free energy (ΔG), also entropy (ΔS). Among the cycloadducts, IC-3 is identified as the greatest thermodynamically stable, exhibiting the lowest ΔG of -8.92 kcal.mol -1 , while IC-5 ranks as the least favorable with the maximum ΔG of -1.35 kcal.mol -1 [ 70 ]. Among the TSs, TS1-en displays the lowest ΔG, indicating it is the greatest kinetically favorable reaction path. It also has the highest GEDT value, suggesting a strong polar character and efficient electron transfer between the reactants. TS1-ex is slightly higher in energy and has a comparable GEDT value, making it the second most favorable. In contrast, TS2-en and TS2-ex exhibit higher ΔG and lower GEDT values, indicating less favorable and less polar TSs. Table 3 Using M06-2X-D3/6-311 + + G(d,p), the 32CA reaction with GEDT stationary points were estimated in toluene at 110°C. The relative energies (∆E), enthalpies (∆H), Gibbs free energies (∆G), also entropies (∆S) kcal.mol -1 were determined using average electron count. Structure ΔE ΔH ΔG ΔS GEDT IC-3 -23.27 -24.21 -8.92 -51.32 ------- IC-4 -21.01 -22.03 -6.27 -52.88 ------- IC-5 -16.39 -17.38 -1.35 -53.79 ------- IC-6 -20.69 -21.69 -5.53 -54.23 ------- TS1-en 13.49 13.07 27.18 -47.35 0.0594 (FEDF) TS1-ex 13.37 12.70 27.94 -51.12 0.0574 (FEDF) TS2-en 14.86 14.15 29.79 -52.49 0.0199 (NEDF) TS2-ex 15.41 14.40 31.61 -57.76 0.0229 (NEDF) The analysis of thermodynamic parameters reinforces that ortho -TS1-en reaction path is the utmost favorable route, both thermodynamically and kinetically (Fig. 3 ). This result supports experimental findings [ 23 ]. The GEDT values reflect the extent of electron transfer, with TS1-en exhibiting the highest GEDT value (0.0594), indicating a tendency toward a polar reaction and classified as FEDF, suggesting a significant electron density flux. TS2-en has a GEDT value of 0.0199, indicating a preference for a non-polar reaction and categorized as NEDF [ 65 , 71 ]. A representation of the optimized structures of the TSs via bond distances of O1–C5 and C3–C4 is shown in Fig. 4 , highlighting the asynchronicity in the 32CA reaction path. In the TS1-en structure, the O1–C5 bond distance is 2.16 Å, however the C3–C4 bond is 2.08 Å, indicating a small change among the two forming bonds. However, in TS1-ex , the O1–C5 bond is 2.03 Å and C3–C4 bond is 2.14 Å, showing a larger difference in bond lengths. The greater disparity in TS1-ex suggests a more asynchronous bond formation compared to TS1-en , where the bond formation is more synchronous. Therefore, TS1-ex is more asynchronous than TS1-en based on the bond distance differences [ 72 ]. How synchronous the structure is (Δd, in Å) is measured by comparing lengths of the two forming sigma bonds. Moreover, in the case of TS1-ex , a Δd of 0.11 Å between the C3–C4 with O1–C5 bonds indicates an asynchronous bond formation, though the bonds form nearly simultaneously. Additional Δd values for other TSs are provided in Table S1 for comparison. 3.4 Analysis of the Topological Shape of ELF at TSs Topological analysis provides insights into electronic structures of optimal TSs participating in 32CA reaction between nitrone GN- 1 and CA-2 . The examination of their ELF analyzed the electrical structures of the TSs. Otherwise, Fig. 5 demonstrates basin attractor positions with ELF localization zones for 32CA reactions' TSs. However, this analysis emphasizes bonding interactions among O1-C5 with C3-C4 in the ortho reaction path, as well as O1-C4 with C3-C5 in the meta reaction path [ 73 ]. The ELF investigation of four reaction paths in 32CA reaction involving nitrone GN-1 with CA-2 reveals V(O1) also V′(O1) basins populations of 5.75, 5.67, 5.85, besides 5.78e for TS1-ex , TS1-en , TS2-ex , with TS2-en , corresponding to non-bonding electron density on O1 (Fig. 4 ). Admittedly, according to the ELF, in the region where N2-C3 bonds are formed, a V(N2,C3) disynaptic basin has been identified. Notably, the N2–C3 bond region has 3.68 electrons in the nitrone GN-1 , but this decreases to 2.67, 2.57, 2.51, and 2.32 electrons in TS2-en , TS2-ex , TS1-ex , and TS1-en , respectively. Through this reduction, a pseudoradical center was produced at carbon atom C3, non-bonding electron density is produced at the nitrogen atom N2. Furthermore, ELF analysis also finds additional disynaptic basin, V(O1,N2), spanning 1.20-1.31e, indicating a single O1–N2 bond in the nitrone GN-1 framework. In addition, each TS also features a disynaptic basin, V(C4,C5), which has a charge distribution of 2.78-2.70e, corresponds to the C4–C5 bond in the CA-2 framework. However, TS1-ex, TS1-en, TS2-ex , with TS2-en indicate a monosynaptic basin V(C3) with values 0.79, 0.48, 0.27, and 0.34e, linked to the C3 carbon atom (see Fig. 5 ). However, TS1-en , monosynaptic basins on C3 with C4 have populations of 0.48 with 0.33 e, respectively, indicating developing or partial lone pair character. The N2 lone pair shows a population of 1.21 electrons. Moreover, in TS1-ex , the monosynaptic basins on C3 and C4 each possess values of 0.79 e, whereas N2 maintains 1.16 e, indicating a more asynchronous bonding process and greater lone pair contribution from the carbon atoms. In TS2-en , C3 and C5 have lower monosynaptic populations of 0.34 and 0.35 electrons, and N2 has 0.88. TS2-ex , the monosynaptic populations on C3 and C5 are 0.27 and 0.29 electrons, and N2 has 1.17 electrons, indicating slightly weaker bonding interactions and partial electron localization [ 74 ]. In contrast, all four competitive reaction paths are defined by the nonappearance of novel disynaptic basins linked to the anew established bonds C3–C4/O1–C5 and C3–C5/O1–C4 for the ortho with meta reaction paths, respectively. However, this indicates the TSs have not yet formed two new covalent connections. The NCI analysis and QTAIM topological analysis study in Section 3.7 also supports this observation. 3.5 How the 32CA reaction works along the different reaction paths, as explained by the Bonding Evolution Theory (BET) BET, developed by Krokoidis, predicts chemical reaction processes [ 75 , 76 ]. Within the 32CA reaction between nitrone GN-1 with CA-2 , the MEDT employed BET to investigate how electron density varies throughout the reaction path. This comprehensive analysis of the bonding pattern along the ortho -regioisomeric pathways identified six ELF topological phases. In general, Figs. 6 , Figs. 7 , Scheme 3 , also Table 4 demonstrate the ELF attractor positions for six Structural Stability Domains (SSDs) ( SSD-1 to SSD-VI ) determined along the IRC connected to the TS1-en of the 32CA reaction path. Meanwhile, this method emphasizes O1-C5 and C3-C4 bonds. Certainly, TS1-en reaction paths' electron density varies through bond creation and breaking, as seen by ELF attractors, resulting in the creation of the 32CA product. However, the step-by-step development and evolution of ELF attractors within structures SSD-1 through SSD-VI indicate the process of bond creation and the stabilization of the product structure. Table 4 In order to determine ( TS1-en ) reaction pathway of the 32CA reaction, which incorporates nitrone GN-1 with CA-2 , ELF valence basin populations of the IRC structures SSD-1 to SSD-VI are crucial. Phases I II III IV V VI structure SSD SSD SSD (TS) SSD SSD SSD d(O1-C5) 3.04 2.16 2.12 1.91 1.57 1.44 d(C3-C4) 3.19 2.13 2.08 1.82 1.62 1.55 GEDT 0.02 0.073 0.057 0.089 0.252 0.307 V(O) 3.02 2.92 2.89 2.89 2.65 2.52 V'(O) 2.91 2.87 2.78 2.82 2.61 2.38 V(O1,N2) 1.40 1.32 1.31 1.15 0.97 0.96 V(N2,C3) 3.68 2.43 2.34 1.97 1.80 1.72 V(C4,C5) 3.26 3.12 2.77 2.27 2.01 1.94 V(C3) 0.37 0.33 V(C4) 0.48 V(C3,C4) 1.45 1.77 1.84 V(N2) 1.10 1.21 1.78 2.15 2.26 V(C5) 0.17 V(O1,C5) 0.93 1.29 Table 4 shows that the electron density of the TS1-en reaction path fluctuates during bond formation and cleavage, as shown by ELF attractors, which are important for understanding the reaction path of 32CA reaction involving nitrone GN-1 and CA-2 . Correspondingly, the table monitors changes in bond distances (such as d(O1-C5), d(C3-C4)), GEDT, and ELF populations throughout different stages of the reaction. Importantly, as the reaction moves forward, bond distances decrease (for instance, O1-C5 declines from 3.04 to 1.44), indicating the formation of bonds. The GEDT values increase, suggesting enhanced electron transfer. ELF populations (like V(N2,C3), V(C4,C5)) demonstrate shifts in electron density, with certain basins emerging or disappearing, illustrating the reorganization of electrons during the reaction [ 65 ]. Figure 6 illustrates the progression of bond creation beside the reaction path of TS1-en in the 32CA reaction among nitrone GN-1 with CA-2 . However, the figure highlights the particular points along the IRC for each SSD structure, emphasizing the evolution of key interactions during the reaction. The depiction illustrates the involvement of atoms O1, C3, C4, N2, and C5 at various stages of the reaction, with specific valence basins corresponding to different phases. The appearance and disappearance of these valence basins indicate significant electronic rearrangements. The presence of V(C3) in the early structures suggests an initial localisation of electron density at C3, which later transforms as the reaction progresses. The emergence of V(C3,C4) also V(O1,C5) in later stages indicates the making of new C–C with O–C bonds. Whenever V(C4) and V(N2) also appear at specific stages, contributing to the reaction mechanism. The red ellipse in the figure highlights the regions where topological alterations occur, marking critical points of electronic reorganization. These changes confirm the gradual bond creation process alongside the reaction coordinate, supporting the transition from reactants to products. The figure effectively captures the evolution of electron density and bonding interactions, providing a visual representation of the reaction mechanism. Six distinct topological phases along the TS1-en chemical pathway were found in ELF basins. The initial SSD-I ELF structure mirrors the bonding forms in the compounds. Moreover, as shown in Fig. 7 , ELF analysis at TS1-en indicates a V(N1) monosynaptic basin, containing approximately 1.1 electrons, at SSD-II , which corresponds to the lone pair on N2 nitrogen. In addition, pseudoradical centers are also found at carbon atom C3 within same SSD-II , thus leading to the development of monosynaptic basin V(C3). However, pseudoradical centers identified at C4 carbon atoms in SSD-III , the monosynaptic basin V(C4) significant contributor to its composition. In general, TS1-en is part of SSD-III , as illustrated in Fig. 7 . However, the O1–C5 with C3–C4 single bonds have not yet been established. Frequently, in SSD-IV , first single bonds occur among C3 with C4. Additionally, in this phase, SSD-IV's C5 carbon atoms have pseudoradical centers, creating the monosynaptic basin V(C5). A new bond is formed between O1 and C5 in SSD-V [ 77 , 78 ]. Scheme 3 illustrates the key electronic and structural changes during the 32CA reaction among nitrone GN-1 and CA-2 , focusing on the TS1-en reaction path. It highlights sequential bonding modifications, such as the shortening of bond distances (e.g., V(C4,C5) decreases from 3.26 to 2.01) and shifts in electron density (e.g., V(N2,C3) drops from 3.68 to 1.8), indicating bond formation and reorganization. The GEDT values increase as the reaction progresses, reflecting enhanced electron transfer. New basins like V(C3,C4) with V(O1,C5) emerge in later stages, signifying the creation of new bonds. The TS ( SSD-III ) is marked by intermediate values, confirming its role as the critical point in the reaction. Overall, the scheme captures the dynamic evolution of electron populations and bonding interactions along the reaction path [ 77 , 78 ]. 3.6 NCI analysis NCI are crucial in influencing the stability with reactivity of molecular systems [ 79 , 80 ]. The NCI analysis was performed on the TS structures ( TS1-ex , TS1-en , TS2-ex , with TS2-en ) to verify existence with significance of these interactions in stabilizing the TSs (Fig. 8 ). NCI gradient isosurfaces reveal weak intermolecular forces for example Van der Waals interactions, hydrogen bonds, aslo steric effects repulsions. Significantly, Fig. 9 demonstrations the four TS scatter plots. However, the RDG for λ2 > 0 and λ2 < 0 identifies dominating interactions based on (λ2)ρ sign. Therefore, the scatter plot of TS1-en and TS2-ex shows a blue region (-0.05 to -0.02 a.u.), thin green band, with a red zone. Usually, for the other TSs, the blue region gets smaller, and the red region becomes a bit smaller as the green region expands [ 81 ]. A significant attraction (blue) exists among O1 with C3 atoms of nitrone GN-1 also C-C atoms (double bonds) of CA-2 across TSs, as seen in Fig. 9 . 3.7 QTAIM topological analysis A QTAIM topological analysis of electron density ρ at critical points (CPs) in the molecular region involved in forming new C-C and O-C single bonds at TSs was conducted to understand better atomic interactions during the 32CA reaction between nitrone GN-1 and CA-2 (Fig. 10 ). The QTAIM variables are in Table 5 . The newly selected critical points, CP1 and CP2, have low electron density ρ(r) values (below 0.1 a.u.) at the four TSs. The small positive ∇²ρr indicates no covalent bonding in these locations. Therefore, creation of new C–C with O–C bonds hasn't started at the TS, matching ELF study results. (see Fig. 10 ). For O1-C5, the electron density is highest in TS1-ex (0.0693 a.u) and lowest in TS1-en (0.0591 a.u), while the Laplacian values follow a similar trend. For C3-C4, TS1-en shows the highest electron density (0.0751 a.u), with TS2-ex has lowest (0.0595 a.u). The Laplacian for C3-C5 is highest in TS2-ex (0.0405 a.u) and lowest in TS1-en (0.0209 a.u). These values indicate variations in bond strength and character across the TSs [ 82 ]. This analysis further highlights the asynchronicity shown by the distance progress indexes discussed in Section 3.5. Table 5 nitrone GN-1 with CA-2 undergo a 32CA reaction. This reaction is affected by the entire electron density ρ (a.u.) also Laplacian of the electron density ∇²ρ(rc), (a.u.), at critical points in the TSs. TSs CP1(O1-C5 a /C4 b ) CP2 (C3-C4 a /C5 b ) 𝝆 𝛁 𝟐 𝝆 𝝆 𝛁 𝟐 𝝆 TS1-en 0.0591 0.119 0.0751 0.0209 TS1-ex 0.0693 0.132 0.0661 0.0301 TS2-en 0.0644 0.124 0.0610 0.0350 TS2-ex 0.0634 0.121 0.0595 0.0405 a for ortho and b for meta . 3.8 Molecular docking study against PDB ID: 2ITZ Molecular docking simulations predict the optimal way a ligand binds to a macromolecular target. They create numerous postures at protein's binding position. Furthermore, best ligand conformations were chosen depending on optimum organization and the lowest free binding energy [ 83 , 84 ]. Additionally, Flare 9 was utilized for molecular docking investigations, and conventional methods and methodology were adhered to. Moreover, using this method, molecules IC-3 , IC-4 , IC-5 , with IC-6 were attached to the active site of the EGFR L858R mutation, in combination with Gefitinib (PDB ID: 2ITZ). This entry showcases the EGFR kinase domain harboring the L858R mutation, a common alteration in lung cancer, in complex with the drug gefitinib (Iressa). Such mutations can affect drug binding and influence treatment efficacy [ 24 ]. Figure 11 illustrates the comparison between the original and re-docked poses of a ligand-protein complex The RMSD value of 1.439 Å (angstroms) measures the difference between the two positions. RMSD (Root Mean Square Deviation) is a common metric used to assess how accurately a docking method can replicate the experimentally established position of a ligand [ 85 ]. Table 6 presents the binding affinities of four molecules IC-3, IC-4, IC-5 , and IC-6 along with the co-crystal ligand, measured in kcal.mol − 1 against the 2ITZ target. However, amongst the established compounds, IC-6 shows the highest binding affinity at -10.19 kcal.mol − 1 , followed by IC-4 and IC-5 . IC-3 has the weakest binding among the new compounds. The co-crystal ligand demonstrates the highest binding affinity overall at -11.43 kcal.mol − 1 , serving as a benchmark for comparison. These results suggest that IC-6 is the most promising candidate among the tested compounds [ 86 ]. Table 6 kcal.mol -1 values indicate the binding affinity of compounds IC-3 , IC-4, IC5 , and IC-6 to the 2ITZ, as well as the co-crystal ligands. Compound Affinity (kcal.mol − 1 ) IC-3 IC-4 IC-5 IC-6 Co-crystal ligand -8.33 -9.63 -9.23 -10.19 -11.43 Figure 12 illustrates the interaction of molecules IC-3 , IC-4 , IC-5, with IC-6 with the EGFR L858R mutant (PDB ID: 2ITZ) using both 3D and 2D representations. The 3D visualization offers a spatial perspective on how these compounds fit within the binding site, displaying molecular conformations and potential interactions such as hydrogen bonding, hydrophobic interactions, or electrostatic forces [87, 88]. The 2D representation highlights specific molecular interactions among the compounds also amino acid residues of target, simplifying the analysis of binding mechanisms. This combined visualization approach facilitates an understanding of how effectively these compounds bind to and interact with the target protein. Table S2 provides a summary of the binding affinities and nonbonding interactions among the 2ITZ target, molecules IC-3 , IC-4 , IC-5 , IC-6 , with co-crystallized ligand. 3.9 Evaluation of drug-likeness and ADMET predictions An ADMET revision assesses a drug's pharmacokinetics by analyzing Absorption, Distribution, Metabolism, Excretion, with Toxicity. Meanwhile, it predicts how a drug behaves also its effects in the body, especially regarding oral also intestinal absorption, which is crucial in drug detection. However, unfortunate absorption can hinder distribution and metabolism, principal to risks like neurotoxicity and nephrotoxicity. Additionally, Table 7 shows the SwissADME-predicted drug-likeness properties for molecules IC-3 , IC-4 , IC-5 , with IC-6 . All four compounds have the same molecular weight (369.16 g/mol), fall within the optimal range, and share identical values for number of hydrogen donors (nHD) (1), hydrogen bond acceptors (nHA) (6), Number of rotatable bonds (nROT) (8), and topological polar surface area (TPSA) (76.07 Ų). Their LogP values, indicating lipophilicity, are also within the favorable range, with IC-4 being the most lipophilic. All compounds fully adhere to Lipinski’s Rule of Five, indicating favorable potential for oral bioavailability. These results indicate that each compound possesses favorable drug-like properties [78]. Hence, these findings demonstrate that all three substances possess features that are similar to those of drugs, making them strong candidates for additional progress in drug finding. Their dependable adherence to Lipinski's rules is especially promising for their potential as drug applicants. Table 7. SwissADME calculated drug-likeness for IC-3 , IC-4 , IC-5 , and IC-6 . Property IC-3 IC-4 IC-5 IC-6 Comments MW 369.16 369.16 369.16 369.16 Contain hydrogen atoms. Optimal:100~600 nHD 1.0 1.0 1.0 1.0 Number of hydrogen bond donors. Optimal:0~7 nROT 8.0 8.0 8.0 8.0 Number of rotatable bonds. Optimal:0~11 LogP 2.118 2.538 2.223 1.766 The logarithm of the n-octanol/water distribution coefficients at pH=7.4. nHA 6.0 6.0 6.0 6.0 Number of hydrogen bond acceptors. Optimal:0~12 TPSA 76.07 76.07 76.07 76.07 Topological Polar Surface Area. Optimal:0~140 Lipinski rule 0.0 (Accepted) 0.0 (Accepted) 0.0 (Accepted) 0.0 (Accepted) MW 500; logP 5; Hacc 10; Hdon 5 n If two properties are out of range, a poor absorption or permeability is possible; one is acceptable. Abbreviations : MW, Molecular weight; nHD, number of hydrogen donors; nROT, Number of rotatable bonds; LogP, Log of the octanol/water partition coefficient; nHA, number of hydrogen acceptors; TPSA, topological polar surface area; The boiled egg model, shown in Figure 13, estimates chemical component absorption in the gastrointestinal system with blood-brain barrier crossing based on WLOGP against TPSA reference plot locations. However, this illustration shows that the yolk implies brain penetration and white region suggests strong passive absorption in gastrointestinal system. The positioning of the compounds within these regions determines their pharmacokinetic behavior. If a compound is located within the yolk, it suggests a high likelihood of crossing the blood-brain barrier, thereby increasing its potential to reach central nervous system. If it is located in the white region, it is more likely to be well absorbed in the intestinal tract, but not necessarily to penetrate the brain. The presence of IC-3 , IC-4 , IC-5 with IC-6 within yolk indicates that these compounds are expected to cross blood-brain barrier (BBB) successfully. Particularly, this prediction is significant for calculating the potential activity of the compounds within the central nervous system [89, 90]. Figure 14 presents a physicochemical radar chart that visually represents the key molecular properties of the selected compounds. This chart provides an overview of multiple parameters simultaneously, offering insights into the balance of different physicochemical characteristics. Each axis of the radar chart corresponds to a specific property, such as lipophilicity, polarity, solubility, molecular weight, flexibility, and saturation. Table S3 summarizes the SwissADME-predicted distribution and excretion properties of molecules IC-3 , IC-4 , IC-5 , with IC-6 . As shown in Table S3, the BBB permeability values for molecules IC-3 , IC-4 , IC-5 with IC-6 are 0.084, 0.206, 0.066, and 0.396, respectively, indicating that compound IC-6 has the highest potential to cross the blood-brain barrier. However, IC-5 shows the lowest. Regarding human liver microsomal (HLM) stability, IC-4 and IC-5 are likely to be less stable, with values closer to 1 indicating a higher probability of metabolic instability. For plasma clearance (CL), all compounds fall within the moderate clearance range (5–15 ml/min/kg), with IC-6 having the highest predicted clearance. These results provide insight into the compounds’ pharmacokinetic behaviors[74]. Table S4 shows SwissADME-based drug-likeness predictions for four molecules IC-3 , IC-4 , IC-5 , with IC-6 , emphasizing their relations with cytochrome-P (CYP) enzymes. However, Table S5 shows the ADMET/pharmacokinetic properties of the best-hit compounds, focusing on key metrics such as permeability, Pgp interactions, and bioavailability. Table S6 summarizes the predicted toxicity profiles of molecules IC-3 , IC-4 , IC-5, with IC-6 , evaluated utilizing OSIRIS Property Explorer and PreADMET. 3.10 Evaluation of antibacterial activity utilizing PASS for the examined substances The antimicrobial activity of compound IC-3 was assessed using PASS [91] . Moreover, the PASS tool provides a simple and efficient method for estimating a compound's potential biological activities [92, 93]. Additionally, it facilitates the early assessment of a compound's pharmacological profile, encompassing therapeutic benefits, mechanisms of action, and safety aspects, including toxicity and side effects. However, the findings are encapsulated in Tables 8 and S7. Moreover, the PASS prediction outcomes show that IC-3 has strong pharmacological potential, as all compounds show activity probabilities (Pa) above 0.7. However, Table 8 shows that IC-3 has a great probability of acting as an aldose reductase substrate, with a probability of activity (Pa) value of 0.842. The probability of inactivity (Pi) is very low at 0.002. This indicates strong confidence that IC-3 may interact with or be metabolised by aldose reductase, suggesting potential relevance in biological or pharmacological contexts involving this enzyme [94, 95]. Table S7 lists the predicted biological activities of the IC-3 compound, showing multiple potential enzyme interactions and pharmacological effects based on PASS analysis with a threshold of Pa > 0.3. The high probabilities for targets such as CDP-glycerol glycerophosphotransferase and various dehydrogenases highlight IC-3 ’s potential multifunctional bioactivity, which may be valuable in drug discovery or biochemical reaction path modulation. Table 8 . PASS prediction of IC-3 compound's main potential activities. The results indicate the probability of a molecule being active, with Pa > 0.7. Biological Activity Pa Pi Aldose reductase substrate 0.842 0.002 Pa = Probability to be active, Pi = Probability to be inactive. 3.11 Molecular Dynamics stimulation The compound IC-6 which showed the best binding affinity against the target proteins (EGFR kinase domain harboring the L858R mutation), were further considered for MD simulation study using Gromacs-2025 software on a GPU system [52]. The protein-ligand complexes generated from the binding affinity prediction analysis were subjected to MD simulation study along with the original crystal structure of the target protein complexed with the co-crystal inhibitors. The protein-ligand complexes were initially cleaned and prepared using UCSF Chimera 1.18. The energy minimization process was performed using the steepest descent method, and Na + and Cl - ions at the concentration of 0.1M were added for neutralization. All essential topology files, including CHARMM parameter files, were generated using the SwissParam server [96]. MD simulations of the complex were conducted for a total simulation time of 100 nanoseconds (ns), collecting snapshots at intervals of 100 picoseconds (ps) from 0 ns to 100 ns at 1 bar and 300 K reference pressure and temperature. The root mean square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) were analyzed for the target proteins (EGFR kinase domain harboring the L858R mutation) and complex forms to test the conformational stability. Figure 15 presents three important MD analysis plots for the nonstructural protein–ligand complex ( IC-6-2ITZ ): RMSD, RMSF, and Radius of Gyration (Rg), each offering insight into the structural stability, flexibility, and compactness of the complex during the simulation period. The root mean square deviation (RMSD) plot (A) illustrates the root mean square deviation of the protein–ligand complex backbone atoms over time, reflecting the structural stability of the complex. A low and relatively constant RMSD curve suggests that the complex remains stable throughout the simulation, with no major conformational changes. If the RMSD increases sharply or fluctuates heavily, this would indicate instability or significant structural rearrangements. The structural changes of the IC-6-2ITZ complex were examined using RMSD. The conformational change of Ligand atoms was estimated and compared to the initial conformation over 100 ns of MD simulations. As can be seen in Figure 15 (A), the overall stability of the investigated complex was observed with a protein’s RMSD value ranging from 0.3 to 1.0 nm. Its fluctuations towards the end of the simulation are around some thermal average structure. From about 60 ns to the end of the simulation time, the RMSD values of the protein stabilize at around 0.75 nm, indicating that the simulation converges, then the system has reached an equilibrium state. Furthermore, Backbone RMSD (red color) indicates how stable the Backbone is with respect to the protein and its binding pocket. At the end of the simulation time, the RMSD value of the Backbone is 0.25 nm, lower than that of ligand by 0.5. Therefore, it is certain that the ligand has not diffused away from its initial binding site. Overall, these results confirmed that the IC-6 inhibitor is tightly bonded and does not affect the overall topology of EGFR. The RMSF plot (B) shows the root mean square fluctuation of each residue, indicating the flexibility of individual amino acids. Higher RMSF values correspond to regions with greater flexibility, such as loops or terminal ends, while lower values are typical of more rigid, structured regions like α-helices or β-sheets. This analysis helps identify which parts of the protein are most dynamic or possibly involved in ligand interactions. The RMSF value for the same complex is mainly below 1 . 3 nm with the strongest fluctuations observed for the residue positions 700 , 722 and 1007 Figure 15 (B). The Radius of gyration (Rg) plot (C) measures the radius of gyration, which provides information about the compactness of the protein structure over time. A consistent Rg value throughout the simulation suggests that the protein maintains its structural integrity and does not undergo significant expansion or contraction. On the other hand, notable changes in the Rg value may indicate partial unfolding or a change in compactness due to ligand binding or conformational drift. Rg values for this complex form a relatively stable profile from 0.37 nm to 0.435 nm Figure 15 (C). 4. Conclusions The study provided deep mechanistic insights into 32CA reaction among glutaraldehyde-N-aryl nitrone with cinnamaldehyde using MEDT. However, the results indicated the reaction adheres to a two-stage, one-step mechanism, aligning with study of bond formation developments. Moreover, global reactivity indices indicated a polar property of process, while nitrone tends to be a strong nucleophile, and cinnamaldehyde is an electrophile. ELF and topological analysis confirmed that the reaction occurs through non-concerted bond formation, validating the stepwise character of the mechanism. Furthermore, GEDT analysis revealed that the reaction pathways are highly asynchronous, showing differences in electron density transfer along regioisomeric channels. Energetic profiles established that the ortho and endo pathways are energetically favored, which aligns with the observed regioselectivity and stereoselectivity. Otherwise, the analysis too confirmed polar nature process plays a decisive role in controlling selectivity, in addition to the mechanistic study, molecular docking, ADMET, with MD simulations used to investigate the biological potential of the resulting cycloadducts. Docking studies suggested strong binding affinities of the products toward biological targets, with favorable pharmacokinetic profiles predicted by ADMET analysis. MD confirmed the stability of ligand–receptor complexes, reinforcing the potential of these molecules as bioactive agents. Overall, the study not only clarified the mechanistic features of the [3+2] cycloaddition but also highlighted the biological relevance and drug-like properties of the synthesized compounds, making them promising candidates for further pharmacological development. Declarations Author contributions: RNS, HMS, and MA calculated and planned the technique. HMS and RNS wrote the manuscript. The authors collectively conducted the discussion and manuscript revision. Funding: The authors declare that they did not receive any awards, cash, or other financial support for writing this work. Conflict of Interest: The writers confirm they have no conflicts of attention. Acknowledgment: The authors express their gratitude to the University of Valencia for given that the essential computing resources for this investigation. However, all calculations presented in this paper were conducted using the Gaussian 16 software on a supercomputer. Additionally, we definite our thankfulness to Cresset for allowing us access to the academic version of Flare V9 Software, located at New Cambridge House, Litlington, Cambridgeshire, UK, for the purpose of conducting molecular docking experiments. Data availability: There was no generation or analysis of any datasets as part of the current investigation. Ethics declaration: not applicable. References Padwa A, Pearson W. Synthesis Applications of 1, 3-Dipolar Cycloaddition Chemistry: Wiley, New York; 1984. Feuer H. Nitrile oxides, nitrones and nitronates in organic synthesis: novel strategies in synthesis: John Wiley & Sons; 2008. Acharjee N, Mohammad-Salim HA, Chakraborty M. 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RMMRs (R k molecular maps of reactivity) for the reaction of nitrone GN-1 and CA-2. > 0 , indicated in red, identifies electrophilic centers, while < 0, shown in blue, indicates nucleophilic centers. scheme3.png Scheme 3. Potential regio- and stereoisomeric reaction paths of 32CA reaction among nitrone GN-1 and CA-2, forming cycloadducts. scheme4.png Scheme 3. TS1-en reaction route sample IRC sites show the 32 CA reaction has successive bonding modifications and the greatest average electron count valence basin populations. which includes nitrone GN-1 and CA-2. 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1","display":"","copyAsset":false,"role":"figure","size":228473,"visible":true,"origin":"","legend":"\u003cp\u003eThe M06-2X-D3/6-311++G(d,p) approach was employed to examine basin attractors of nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e and \u003cstrong\u003eCA-2\u003c/strong\u003e, as well as their ELF domains. The color-coded diagram uses blue to denote protonated basins, red signifies monosynaptic basins, while green represents disynaptic basins, and purple indicates attractor sites with an isovalue of 0.80. Whenever it also shows the mean electron count, atomic charges, and Lewis structures. Blue shows positive charges, and red designates negative ones.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/96942b93beb1535b3d85c31f.png"},{"id":94451707,"identity":"70a86721-d9d4-4ee7-a9ab-1c172c6dccfd","added_by":"auto","created_at":"2025-10-27 14:40:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":248218,"visible":true,"origin":"","legend":"\u003cp\u003eM06-2X-D3/6-311++G(d,p) nucleophilic \u0026nbsp;\u0026nbsp;P\u003csub\u003ek\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e Parr functions of nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e and electrophilic \u0026nbsp;\u0026nbsp;\u0026nbsp;Parr functions of \u003cstrong\u003eCA-2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/7b9f3a930102353012dbb650.png"},{"id":94451507,"identity":"6fd46220-c4da-4ce6-b7f2-5af2ddd41a25","added_by":"auto","created_at":"2025-10-27 14:40:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105371,"visible":true,"origin":"","legend":"\u003cp\u003eM06-2X-D3/6–311G++(d,p) ΔG, in kcal.mol\u003csup\u003e-1\u003c/sup\u003e of the 32CA reaction of the nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e and \u003cstrong\u003eCA-2\u003c/strong\u003e, in toulene at 110 °C.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/2456054cb5df0a8e48dd5488.png"},{"id":94451160,"identity":"ad410359-2d34-4bde-b218-a76f4756e5eb","added_by":"auto","created_at":"2025-10-27 14:39:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268388,"visible":true,"origin":"","legend":"\u003cp\u003eStructure M06-2X-D3/6-311++G(d,p) optimized geometry for TSs in toluene, purple represents attractor positions.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/2667109113afa7793720f0ea.png"},{"id":94489368,"identity":"a7d1bc4f-f3ca-41cc-b08b-f787f6b6e752","added_by":"auto","created_at":"2025-10-27 17:04:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1821074,"visible":true,"origin":"","legend":"\u003cp\u003eThe localization domains for M06-2X-D3/6-311++G(d,p) ELF, along with the positions of the attractor basins \u003cstrong\u003eTS1-en\u003c/strong\u003e, \u003cstrong\u003eTS1-ex\u003c/strong\u003e, \u003cstrong\u003eTS2-en\u003c/strong\u003e, and \u003cstrong\u003eTS2-ex\u003c/strong\u003e, are identified. Whenever monosynaptic basins are red, protonated basins are blue, disynaptic basins are green, with attractors are purple.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/9795f68bbb8273bd8f3b07f1.png"},{"id":94450895,"identity":"08de426d-124e-4fd6-8756-e0c83b7362c9","added_by":"auto","created_at":"2025-10-27 14:39:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247215,"visible":true,"origin":"","legend":"\u003cp\u003eThe selected sites of each \u003cstrong\u003eSSD\u003c/strong\u003e documented throughout IRC were associated with the development of bonds between C3-C4 with O1-C5 along the \u003cstrong\u003eTS1-en\u003c/strong\u003e reaction pathway of the 32CA involving nitrone \u003cstrong\u003eGN-1\u003c/strong\u003eand \u003cstrong\u003eCA-2\u003c/strong\u003e. The red ellipse characterized the topological alterations that occurred.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/54a61eb12a94b187f38f9862.png"},{"id":94451526,"identity":"6fd845bd-d22e-4681-a962-cab4c98c9d66","added_by":"auto","created_at":"2025-10-27 14:40:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":110769,"visible":true,"origin":"","legend":"\u003cp\u003eAt the M06-2X-D3/6-311++G(d,p) level, the IRC profile of \u003cstrong\u003eTS1-en\u003c/strong\u003e for the 32CA reaction of nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e with \u003cstrong\u003eCA-2\u003c/strong\u003eis presented.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/8f9388d54547db6915597737.png"},{"id":94450780,"identity":"10f75fad-315c-4c2f-b945-bf2666b21dc7","added_by":"auto","created_at":"2025-10-27 14:39:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":304262,"visible":true,"origin":"","legend":"\u003cp\u003eM06-2X-D3/6–311++G(d,p) NCI isosurfaces at an isovalue of 0.4, illustrating the density overlap at the TSs included in the 32CA reaction among nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e with \u003cstrong\u003eCA-2\u003c/strong\u003e is -0.02 \u0026lt; sign(λ2) ρ(r) \u0026lt; 0.02 a.u.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/6773e4cdb45756c744e43483.png"},{"id":94451153,"identity":"36c85539-7a2a-45b7-be9c-5a6a2e310f68","added_by":"auto","created_at":"2025-10-27 14:39:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":698510,"visible":true,"origin":"","legend":"\u003cp\u003eLower density gradient with gradient isosurfaces of TSs have a role in the 32CA reaction among nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e with \u003cstrong\u003eCA-2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/fa08ca5984c2d3556278be30.png"},{"id":94489336,"identity":"fa059f0c-ae60-4b9c-85cb-3a1e7d06715a","added_by":"auto","created_at":"2025-10-27 17:04:13","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":289805,"visible":true,"origin":"","legend":"\u003cp\u003eTSs are shown in M06-2X-D3/6–311G++(d,p) AIM form. The yellow ellipses show the bond critical points of newly formed bonds.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/0e8f67f8503a0396f614ba11.png"},{"id":94451835,"identity":"03c98379-fdd0-43fc-a126-57d1b80981c1","added_by":"auto","created_at":"2025-10-27 14:40:26","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":254968,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD value = 1.439 A for the re-docking pose (Yellow = Docked, Blue = Original)\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/9236181238508fd44393f899.png"},{"id":94451482,"identity":"2741466f-f1ed-4a88-a697-cf7fc64dc2f1","added_by":"auto","created_at":"2025-10-27 14:40:10","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":2182661,"visible":true,"origin":"","legend":"\u003cp\u003eThe study examined how chemicals \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e, with \u003cstrong\u003eIC-6\u003c/strong\u003e interacted with EGFR in together three-dimensional (3D) with two-dimensional (2D) contexts L858R (PDB ID: 2ITZ).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/6ea1e2fc6a14c3cccceffe02.png"},{"id":94450896,"identity":"40c132b1-6a0b-46d2-9791-ea005d59afbf","added_by":"auto","created_at":"2025-10-27 14:39:39","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":53997,"visible":true,"origin":"","legend":"\u003cp\u003eChemicals that reach egg yolks can pass the blood-brain barrier.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/9bcee9dac75855a3cc99671d.png"},{"id":94451719,"identity":"46c39428-302c-4c4b-973e-75a43dcac584","added_by":"auto","created_at":"2025-10-27 14:40:19","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":304720,"visible":true,"origin":"","legend":"\u003cp\u003ePhysicochemical radar chart of the selected dataset compounds.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/374547fdd7e80967179fe0db.png"},{"id":94450390,"identity":"d4820cb1-2b96-4a5b-99aa-8534566fad2e","added_by":"auto","created_at":"2025-10-27 14:39:14","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":428390,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD (A), RMSF (B), with Rg (C) analysis profiles of the nonstructural protein (\u003cstrong\u003eIC-6-2ITZ)\u003c/strong\u003e complex.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/2f33a0ac8b94ded26649f9f6.png"},{"id":100614664,"identity":"79aee94f-c076-4648-b3e6-600d8303a7d5","added_by":"auto","created_at":"2026-01-19 17:22:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9565209,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/673db4c2-0b82-4e19-a300-e972972a4687.pdf"},{"id":94451249,"identity":"0ab8e94b-01b1-40a0-9eca-ec0dead855ae","added_by":"auto","created_at":"2025-10-27 14:39:59","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":276480,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/6e898fc7ec73be86d8fb7abb.doc"},{"id":94489126,"identity":"0514683e-e578-4648-b52f-c8e9f6acf8bd","added_by":"auto","created_at":"2025-10-27 17:03:28","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":93275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eThe stereoisomeric reaction paths for nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e with \u003cstrong\u003eCA-2\u003c/strong\u003e 32CA reactions.\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/8b479c3b87f8ccebb84741a5.png"},{"id":94451197,"identity":"96d79a6a-cfd0-48a5-b2e6-157b31fcf6e5","added_by":"auto","created_at":"2025-10-27 14:39:56","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2\u003c/strong\u003e. RMMRs (R\u003csub\u003ek\u003c/sub\u003e \u0026nbsp;molecular maps of reactivity) for the reaction of nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e and \u003cstrong\u003eCA-2\u003c/strong\u003e. \u0026nbsp;\u0026nbsp;\u0026gt; 0 , indicated in red, identifies electrophilic centers, while \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026lt; 0, shown in blue, indicates nucleophilic centers.\u003c/p\u003e","description":"","filename":"scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/9b72d40f5170390d175b7b61.png"},{"id":94451426,"identity":"be105a0d-9ad4-4f52-a848-c757f3b72a5e","added_by":"auto","created_at":"2025-10-27 14:40:07","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":148285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 3\u003c/strong\u003e. Potential regio- and stereoisomeric reaction paths of 32CA reaction among nitrone \u003cstrong\u003eGN-1\u003c/strong\u003e and \u003cstrong\u003eCA-2\u003c/strong\u003e, forming cycloadducts.\u003c/p\u003e","description":"","filename":"scheme3.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/097d1e91faabeafcafdfe362.png"},{"id":94489583,"identity":"3e6039f8-65b6-4d82-a1ca-70a27773a41a","added_by":"auto","created_at":"2025-10-27 17:05:12","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":114725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 3.\u003c/strong\u003e \u003cstrong\u003eTS1-en \u003c/strong\u003ereaction route sample IRC sites show the 32 CA reaction has successive bonding modifications and the greatest average electron count valence basin populations. which includes nitrone \u003cstrong\u003eGN-1 \u003c/strong\u003eand \u003cstrong\u003eCA-2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"scheme4.png","url":"https://assets-eu.researchsquare.com/files/rs-7773346/v1/5d488bd968c814e5e96a40e5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic Insights into (3+2) Cycloaddition of Glutaraldehyde-N-Aryl Nitrone with Cinnamaldehyde: Electron Density, Docking, and Molecular Dynamics Analysis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA three-atom component (TAC) reacts with alkene via 32CA, considered one of the most effective ways to synthesize five-membered heterocycle compounds. Additionally, this reaction can produce cyclic compounds via great regio- and stereoselectivity in organic chemistry synthesis. This reaction can form cyclic compounds with high regio- and stereoselectivity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nitrones serve as important TACs commonly used in 32CA reactions. These compounds produce pharmacologically active isoxazolidines and isoxazolines, distinguished by their stereochemistry regioselectivity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In the last ten years, detailed studies using conceptual density functional theory (DFT) continuously nitrone cycloadditions had continuously revealed a strong relationship between experimental outcomes with DFT-predicted selectivity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, research released post-2003 categorizes TACs and alkenes based on absolute electrophilicity, showing electron density changes during cycloaddition [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, since 2003, numerous pathways for 32CA responses have been explored and suggested. Subsequently, in 2014 and 2017, researchers hypothesized one-step with two-step polar mechanisms for 32CA reactions involving nitrones [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Subsequently, Domingo illustrated the MEDT in 2016, emphasizing how electron density fluctuations influence organic reaction reactivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Particularly, Domingo\u0026rsquo;s MEDT [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] is a crucial framework for grasping reactivity in molecular organic chemistry. Consequently, it demonstrates how fluctuations in electron density drive reactivity. Moreover, MEDT research has elucidated the mechanisms of 32CA reactions, demonstrating that they occur through the sequential alteration of bonds throughout one-step reactions. Additionally, distinguishes them starting the coordinated pericyclic mechanism described by Woodward with Hoffman 1969. Although certain 32CA reactions are non-polar, Parr functions help clarify the regioselectivity seen in polar conditions. Additionally, in 2014, Tang and colleagues conducted a DFT study to examine the reaction between 1,3-dialkynes also ammonia derivatives [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Normally, Jasinski (2018) proposed that in the polar nitrone 32CA reactions via arylacetylenes, both one-step and two-step reactions occur simultaneously, competing with each other [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, new investigations in 2017 and 2018 has shown nitrone 32CA reactions are one-step, two-stage with asynchronous bond development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Generally, a wide range of chemicals were used to come up with a standard way to group TACs into four groups: [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] \u003cem\u003epseudodiradical\u003c/em\u003e (\u003cem\u003epdr\u003c/em\u003e) type [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003epseudo\u003c/em\u003e(\u003cem\u003emono\u003c/em\u003e)\u003cem\u003eradical\u003c/em\u003e (\u003cem\u003epmr\u003c/em\u003e) type, \u003cem\u003ecarbenoid\u003c/em\u003e (\u003cem\u003ecb\u003c/em\u003e) type also \u003cem\u003ezwitterionic\u003c/em\u003e (\u003cem\u003ezw\u003c/em\u003e) type [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, relative reactivity movement goes in the order: pdr type\u0026thinsp;\u0026gt;\u0026thinsp;pmr type\u0026thinsp;\u0026asymp;\u0026thinsp;cb type\u0026thinsp;\u0026gt;\u0026thinsp;zw type. Therefore, MEDT involves understanding reactivity, indicating that shifts in electron density cause molecular changes in reactivity. In addition, A regular of quantum chemical tools, including ELF [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], However, ELF, global and local reactivity indices [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and Laplacian of electron density [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this contribution, the MEDT is employed to analyze the 32CA between Glutaraldehyde-N-aryl nitrone \u003cb\u003eGN-1\u003c/b\u003e and Cinnamaldehyde \u003cb\u003eCA-2\u003c/b\u003e (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, the 32CA reaction remained recent established by Sivadharani and Jayapradha [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] as a novel methodology for producing isoxazolidine, which involves adding nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e in catalyst-free conditions (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A mixture of nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e is refluxed in toluene (50ml) for the time period specified in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After 18 hours, the reaction afforded the major product, (3S,4R,5S)-4-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-5-carbaldehyde (\u003cb\u003eIC-3\u003c/b\u003e), in 89% yield. There were also small amounts of another product seen to form [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThis work objects to demonstrate the energetics also reaction mechanism of the computationally 32CA between nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e. Furthermore, the interactions between molecules (3S,4R,5S)-4-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-5-carbaldehyde (\u003cb\u003eIC-3)\u003c/b\u003e, (3R,4S,5R)-4-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-5-carbaldehyde (\u003cb\u003eIC-4)\u003c/b\u003e, (3S,4R,5S)-5-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-4-carbaldehyde (\u003cb\u003eIC-5)\u003c/b\u003e and (3R,4S,5R)-5-(4-hydroxy-3-methoxyphenyl)-3-(4-oxobutyl)-2-phenylisoxazolidine-4-carbaldehyde (\u003cb\u003eIC-6)\u003c/b\u003e through the \u003cb\u003e2ITZ\u003c/b\u003e structures were estimated via molecular docking simulations. However, EGFR L858R Mutation with Gefitinib Protein Data Bank (PDB ID: \u003cb\u003e2ITZ\u003c/b\u003e): This entry showcases the EGFR kinase domain harboring the L858R mutation, a common alteration in lung cancer, in complex with the drug gefitinib (Iressa). Such mutations can affect drug binding and influence treatment efficacy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, the prediction of ADMET properties are crucial in drug investigation and progress. Consequently, once docking, the ADMET of \u003cb\u003eIC-3\u003c/b\u003e, \u003cb\u003eIC-4\u003c/b\u003e, \u003cb\u003eIC-5\u003c/b\u003e, also \u003cb\u003eIC-6\u003c/b\u003e were assessed and compared with control molecules for each target. Moreover, this evaluation provided a significant understanding of their capabilities for other advancement in medication detection and progress. Altogether, manuscript integrates mechanistic organic chemistry with MD\u0026ndash;based validation to highlight both theoretical insights and therapeutic potential of the synthesized isoxazolidine derivatives.\u003c/p\u003e"},{"header":"2. Computational method","content":"\u003cp\u003eImproved chemical structures were obtained utilizing M06-2X-D3/6-311G++(d, p) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] theoretical level, along with Berny analytical gradient optimization [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, because they didn't have any imaginary frequencies. Moreover, presence of one imaginary frequency identified TSs. However, to account dispersion effects influencing the stability of complexes and intermediates, the D3 correction was useful [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, Geometry optimizations used default convergence criteria for geometry, integration grid, as well as SCF procedures in Gaussian 16. Therefore, open-shell systems which handled utilizing a spin-restricted formalism. However, Intrinsic Reaction Coordinate (IRC) analyzed the whole reaction path. Therefore, this approach enables the identification of the reaction path also intermediate states, beside their associated activation energies. All computational analyses were conducted utilizing Gaussian 16, Revision B.01 [\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, the entire reaction path examined utilizing the IRC methodology. However, this method enables a clearer view of the reaction path and assists in identification of intermediate states, along with their corresponding activation energies [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To characterize the polarity of the reaction, GEDT [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], the TS was found by adding up the natural atomic charges (q), which were found in a natural bond orbital (NBO) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] study of the atoms within a separately framework (f); namely, i.e., \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{G}\\text{E}\\text{D}\\text{T}=\\sum\\:_{i\\in\\:f}{q}_{i}.\\:\\:\\)\u003c/span\u003e\u003c/span\u003eFurthermore, Positive values show electronic flux from the framework to the alternative, conversely. Identically, utilizing the framework of conceptual DFT, various descriptors of electronic structure and reactivity were assessed, incorporating the energies of the highest occupied molecular orbital (HOMO, εH) with lowest unoccupied molecular orbital (LUMO, εL). Conversely, this includes LUMO-HOMO gap (gap\u0026thinsp;=\u0026thinsp;εL - εH), global electrophilicity (ω\u0026thinsp;=\u0026thinsp;\u0026micro;2/2η) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and chemical potential [\u0026micro; = (εH\u0026thinsp;+\u0026thinsp;εL)/2] [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] with the global hardness (η\u0026thinsp;=\u0026thinsp;ε\u003csub\u003eL\u003c/sub\u003e - ε\u003csub\u003eH\u003c/sub\u003e) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Chemical softness (S) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] is the opposite of chemical hardness and indicates susceptibility of a molecule's electron density to distortion or perturbation. However, the nucleophilicity index (N) was determined using the formula N\u0026thinsp;=\u0026thinsp;ε\u003csub\u003eH\u003c/sub\u003e (Nu) \u0026minus; ε\u003csub\u003eH\u003c/sub\u003e (TCE), where Nu represents nucleophile while TCE denotes tetracyanoethylene reference [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nonetheless, neutral species and their radical anions and cations were used in natural population analysis (NPA) to calculate Parr, Pearson, and Yang local indices [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Moreover, atomic spin density (ASD) characterizes these functions to assess polarity reactions. While Parr functions, well-defined as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{k}^{+}\\)\u003c/span\u003e\u003c/span\u003e for electrophilic attack also \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{k}^{-}\\)\u003c/span\u003e\u003c/span\u003e for nucleophilic attack, were expressed relative to the respective population \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{q}_{k}\\:\\)\u003c/span\u003e\u003c/span\u003e(N) of k\u003csup\u003eth\u003c/sup\u003e atomic site of the N-electron system, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{k}^{+}={q}_{k}(N+1)-{q}_{k}\\left(N\\right)\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{k}^{-}={q}_{k}\\left(N\\right)-{q}_{k}(N-1)\\)\u003c/span\u003e\u003c/span\u003e respectively. Similarly, the local electrophilicity, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\omega\\:}_{k}\\)\u003c/span\u003e\u003c/span\u003e, which gained via \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\omega\\:}_{k}=\\:\\omega\\:\\:\\times\\:\\:{P}_{k}^{+}\\)\u003c/span\u003e\u003c/span\u003e, also the local nucleophilicity indices, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{N}_{k}\\)\u003c/span\u003e\u003c/span\u003e, which determined via \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{N}_{k}=N\\times\\:{P}_{k}^{-}\\)\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] for a specific input reaction, Domingo et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] suggested a local reactivity difference index \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{k}\\)\u003c/span\u003e\u003c/span\u003e, illustrated as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{k}={(\\omega\\:}_{k}+{N}_{k})/2\\)\u003c/span\u003e\u003c/span\u003e, utilized to identify the species' greatest electrophilic and nucleophilic centers. However, all computations used Gaussian16. Moreover, Non-Covalent Interaction (NCI) analysis involved performance on each optimized TS geometry and using a single point calculation. Furthermore, the related graphs of electron density [ρ(r)] against the reduced density gradient (RDG) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:s\\left(r\\right)=\\left|{\\nabla\\:}_{\\rho\\:}\\left(r\\right)/2{\\left(3{\\pi\\:}^{2}\\right)}^{1/3}{\\rho\\:\\left(r\\right)}^{4/3}\\right|\\:\\)\u003c/span\u003e\u003c/span\u003ewhich generated utilizing the NCIPLOT software [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, the ELF of the molecule was determined with Topmod [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Above all, the Multiwfn program [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] was employed for the topological study of the ELF besides the Quantum Theory of Atoms in Molecules (QTAIM). In addition, natural population analysis (NPA) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] was done at the TSs to find out what the atoms' natural charges (\u003cem\u003eq\u003c/em\u003e) are. A thorough examination of the BET [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] was done to investigate the bonding transformations throughout experimentally established reaction path, including nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e. Moreover, the ELF domains were pictured via VMD [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Graphical illustrations of the ELF attractor basin placements were created utilizing the GaussView6 program [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In addition, visualization of ELF domains was accomplished with Paraview [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Following optimization, molecules \u003cb\u003eIC-3\u003c/b\u003e, \u003cb\u003eIC-4\u003c/b\u003e, \u003cb\u003eIC-5\u003c/b\u003e and \u003cb\u003eIC-6\u003c/b\u003e were employed for molecular docking studies, which were achieved via Cresset Flare V9 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Moreover, three-dimensional (3D) structures of the molecular targets were acquired from Protein Data Bank (PDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rcsb.org\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, the schematic depiction of the chemical pathways was produced utilizing PerkinElmer ChemDraw. Moreover, forecasts of ADMET characteristics (absorption, distribution, metabolism, excretion, with toxicity) performed for combined drugs utilizing the SwissADME platform also the ADMETlab 3.0 web server. However, A PASS (Prediction of Activity Spectra for Substances) study was conducted to forecast their biological activities. Furthermore, they were subsequently subjected to MD simulations using Gromacs 2025 on a GPU-accelerated system [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eThis paper consists of eleven main sections number: (1) The ELF [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] is looked at to learn more about the electronic structure of nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e when they are in their ground state (GS). (2) Furthermore, CDFT [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] has been used to calculate the polar attributes and electronic distribution of the reactions. (3) Researchers have thoroughly studied the potential energy surface (PES) to discovery stationary sites along the possible reaction paths of the 32CA reactions in the investigation. (4) Moreover, the electronic structure of the identified TSs was examined utilizing ELF topological analysis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and QTAIM [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] parameters. (5) A BET study was conducted along the reaction path. (6) The study of NCI is very significant for understanding how the stability and behavior of molecular systems are affected. (7) An elementary examination of the Atoms-in-Molecules (AIM) at the TSs was conducted. (8) Molecular docking studies examined interactions between cycloadducts \u003cb\u003eIC-3\u003c/b\u003e, \u003cb\u003eIC-4\u003c/b\u003e, \u003cb\u003eIC-5\u003c/b\u003e, with \u003cb\u003eIC-6\u003c/b\u003e (2ITZ). (9) Additionally, the material's drug-like properties were evaluated, along with its ADMET assessment characteristics. (10) PASS analysis is conducted using the PASS online platform. (11) Finally, Molecular dynamics (MD) allows the exploration of stability and dynamics of complex systems. It provides insights into structural stability, flexibility, and interactions of biomolecules under near-physiological conditions.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.1 A study of the ELF topology of nitrone \u003cb\u003eGN-1\u003c/b\u003e with Cinnamaldehyde \u003cb\u003eCA-2\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eBecke and Edgecombe [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] originally formulated the ELF, which Silvi and Savin later enhanced [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While ELF has been established as a precise computational tool for studying the electronic structure of chemical systems processes. However, ELF efficiently delineates electronic regions like core, bonding, with non-bonding areas as attractors. ELF delineates basins of attractors as areas where the likelihood of locating electron pair is maximized. Otherwise, these basins are divided into core basins (C(...)) also valence basins (V(...)). Moreover, valence basins were categorized through their synaptic order, indicating the number of atomic valence shells involved. Nevertheless, it can be monosynaptic, disynaptic, trisynaptic, etc [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Domingo's examination of ELF topology provides key insights for classifying 32CA reaction TACs as \u003cem\u003epseudo-diradical\u003c/em\u003e, \u003cem\u003epseudo(mono)radical\u003c/em\u003e, \u003cem\u003ecarbenoid\u003c/em\u003e, with \u003cem\u003ezwitterionic\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, monosynaptic basins, indicated V(A), represent lone pairs or non-bonding regions. Moreover, disynaptic basins, marked V(A,B), associate the centers of two nuclei, A with B, while showing the bonding area between them. A monosynaptic basin with fewer than one electron is linked to a \u003cem\u003epseudoradical\u003c/em\u003e center [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], while one with more than one electron is linked to a \u003cem\u003ecarbenoid\u003c/em\u003e center. Moreover, this study analyzes electronic structures of nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e involved 32CA reactions using ELF topological analysis. In addition, ELF topology of nitrone \u003cb\u003eGN-1\u003c/b\u003e demonstrations two monosynaptic basins, V(O1) also V\u0026prime;(O1), with 5.92 e, and two disynaptic basins, V(N2,C3), also V(N2,O1), with 3.77 e and 1.38 e. e, respectively. Furthermore, monosynaptic basins V(O1) also V\u0026prime;(O1) denote the nonbonding electron density associated with the O1 oxygen atom. Similar to this, underpopulated N2-C3 double bond is linked to disynaptic basin V(N2,C3), whereas underpopulated N2\u0026ndash;O1 single bond is linked to V(N2,O1). Consequently, nitrone \u003cb\u003eGN-1\u003c/b\u003e can be able to categorized as a \u003cem\u003ezwitterionic\u003c/em\u003e TAC because of deficiency of \u003cem\u003epseudoradical\u003c/em\u003e or \u003cem\u003ecarbenoid\u003c/em\u003e centers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMoreover, ELF of \u003cb\u003eCA-2\u003c/b\u003e displays a V(C4,C5) disynaptic basin that integrates 3.30 electrons and is associated to the ethylene C4 and C5 atoms' double bonding area. In addition, the ELF valence basin populations represent the electron localisation in different molecular regions, helping to understand bonding characteristics and electronic structure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsequently, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrations Lewis-like structures of nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e reactants, with atomic charges from NBO analysis. In \u003cb\u003eGN-1\u003c/b\u003e, oxygen (O1) has a charge of \u0026minus;\u0026thinsp;0.61 e, nitrogen (N2)\u0026thinsp;+\u0026thinsp;0.059 e, carbon (C3)\u0026thinsp;+\u0026thinsp;0.073 e. In \u003cb\u003eCA-2\u003c/b\u003e, carbon atoms take charges of \u0026minus;\u0026thinsp;0.11 e and \u0026minus;\u0026thinsp;0.32 [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Analysis of CDFT Indices for Nitrone \u003cb\u003eGN-1\u003c/b\u003e with Cinnamaldehyde \u003cb\u003eCA-2\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eAn examination of the CDFT [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] indices offers a preliminary insight into the reactivity of organic compounds in polar processes. Admittedly, concept of \"Conceptual DFT,\" introduced by Parr, has been widely utilized to evaluate the chemical reactivity of structures included in 32CA reactions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. For the CDFT investigation, Domingo's M06-2X-D3/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p) standard scales for electrophilicity and nucleophilicity indices were employed [\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Using M06-2X-D3/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p) level of theory, CDFT indices for nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e calculated based on established reactivity scales. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Similarly, the chemical potential (\u0026micro;), chemical hardness (η), chemical softness (S), electrophilicity (ω), also nucleophilicity (N) expressed in electron volts (eV) units. Moreover, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicates that chemical hardness (η) for nitrone \u003cb\u003eGN-1\u003c/b\u003e is about 7.11 eV. However, electronic chemical potential (\u0026micro;) of nitrone \u003cb\u003eGN-1\u003c/b\u003e is \u0026minus;\u0026thinsp;4.24 eV, upper that of \u003cb\u003eCA-2\u003c/b\u003e. The calculated value for \u003cb\u003eCA-2\u003c/b\u003e is \u0026minus;\u0026thinsp;4.34 eV. Overall, \u003cb\u003eCA-2\u003c/b\u003e has the highest electronic chemical potential. Additionally, the following is a record of chemical softness (S) values that were determined for the reactants: nitrone \u003cb\u003eGN-1\u003c/b\u003e has a value of 0.14 eV, whereas \u003cb\u003eCA-2\u003c/b\u003e exhibits the highest value at 0.16 eV. On the other hand, nitrone \u003cb\u003eGN-1\u003c/b\u003e takes an electrophilicity (ω) index [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] of 1.27 eV, categorizing it as a moderate electrophile based on electrophilicity scale [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Additionally, chemical softness (S) values for reactants show nitrone \u003cb\u003eGN-1\u003c/b\u003e at 0.14 eV and \u003cb\u003eCA-2\u003c/b\u003e at 0.16 eV, the highest. Nitrone \u003cb\u003eGN-1's\u003c/b\u003e electrophilicity (ω) index is 1.27 eV [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] making it a moderate electrophile per electrophilicity scale [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Furthermore, nucleophilicity index of this substance is 3.11 eV, which indicates that it is a strong nucleophile according to nucleophilicity scale [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Overall, the electrophilicity (ω) index of \u003cb\u003eCA-2\u003c/b\u003e was 1.53, reflecting its strong electrophilic character. Notably, nitrone \u003cb\u003eGN-1\u003c/b\u003e shows higher nucleophilicity than \u003cb\u003eCA-2\u003c/b\u003e, indicating a greater propensity to function as a nucleophile. The GEDT [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] will occur from nitrone \u003cb\u003eGN-1\u003c/b\u003e to \u003cb\u003eCA-2\u003c/b\u003e. Consequently, it is expected that the electronic flux goes among nitrone \u003cb\u003eGN-1\u003c/b\u003e to \u003cb\u003eCA-2\u003c/b\u003e, which is in accordance with the findings on electronic chemical potential (\u0026micro;). Additionally, values of (η) support the nucleophilic nature nitrone \u003cb\u003eGN-1\u003c/b\u003e with the electrophilic nature of \u003cb\u003eCA-2\u003c/b\u003e. However, 32CA reaction demonstrates clearly polar behavior, termed forward electron density flux (FEDF), resulting from nitrone \u003cb\u003eGN-1\u003c/b\u003e's strong nucleophilicity and the significant electrophilicity of \u003cb\u003eCA-2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCalculations at M06-2X-D3/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p) level provided ground-state electronic properties of nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e, including chemical hardness η, chemical potential \u0026micro;, chemical softness S, global electrophilicity ω, with global nucleophilicity N, each shown in eV.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReagent\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eη\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026micro;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eω\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGN-1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCA-2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFurthermore, when two species with different properties interact in a polar 32CA reaction, the preferred pathway is typically determined by the interaction between the location that is the most electrophilic for one species and the site that is the most nucleophilic for the other species [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Nevertheless, several studies emphasize the significance of electrophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e) with nucleophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e) Parr functions [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Using additional spin electron density from GEDT, these functions are one of the best dependable ways to evaluate local reactivity in polar also ionic systems processes. However, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows Parr nucleophilic ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e ) also electrophilic ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e ) functions, local electrophilic (ωk), nucleophilic (Nk) indices, the local reactivity difference index (R\u003csub\u003ek\u003c/sub\u003e). In addition, examination of electrophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e) with nucleophilic ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e ) Parr functions in nitrone \u003cb\u003eGN-1\u003c/b\u003e shows that C3 carbon atom is the greatest electrophilic site, exhibiting ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e )\u0026thinsp;=\u0026thinsp;0.37, whilst the least nucleophilic site is O1, with ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e ) = -0.031. In the instance of \u003cb\u003eCA-2\u003c/b\u003e, the C4 is designated as least electrophilic site ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e = 0.25), whereas the most nucleophilic site is also C4 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e= 0.038) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The \u003cem\u003eR\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e values show the most favorable interactions between electrophiles and nucleophiles along the reaction pathway. It was found that the main interaction in this condensation occurs among the electrophilic center, C5 (\u003cem\u003eR\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e= 0.23) of \u003cb\u003eCA-2\u003c/b\u003e, nucleophilic center, O1 (\u003cem\u003eR\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e = -0.038) of nitrone \u003cb\u003eGN-1\u003c/b\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the 3D representation of the nucleophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e) also electrophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e) Parr functions between nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThere are local electrophilic and nucleophilic indices (ωk, Nk) with local reactivity difference indices (\u003cem\u003eR\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e) values for M06-2X-D3/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p). These include nucleophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e) with electrophilic (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e) Parr functions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReactant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCenter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{+}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{\\text{k}}^{-}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eω\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003ek\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eGN-1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e-0.32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-0.99\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-0.38\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.47\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eCA-2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.038\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.38\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.093\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eGN-1 CA-2\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eScheme 2\u003c/b\u003e. RMMRs (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{R}}_{\\text{k}}\\)\u003c/span\u003e\u003c/span\u003e molecular maps of reactivity) for the reaction of nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{R}}_{\\text{k}}\\)\u003c/span\u003e\u003c/span\u003e\u0026gt;0, indicated in red, identifies electrophilic centers, while \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{R}}_{\\text{k}}\\)\u003c/span\u003e\u003c/span\u003e \u0026lt; 0, shown in blue, indicates nucleophilic centers.\u003c/p\u003e\u003cp\u003eHowever, Chattaraj introduced local reactivity modification index in 2012 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], a tool for predicting the local electrophilic aslo nucleophilic activation of organic compound molecules [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In Scheme 2, nitrone \u003cb\u003eGN-1\u003c/b\u003e shows \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{R}}_{\\text{k}}\\)\u003c/span\u003e\u003c/span\u003e values of -0.38 for O1 and \u0026minus;\u0026thinsp;0.15 for C3, indicating a tendency toward nucleophilicity. Conversely, for \u003cb\u003eCA-2\u003c/b\u003e, the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{R}}_{\\text{k}}\\)\u003c/span\u003e\u003c/span\u003e values of C4 also C5 are 0.17 and 0.23, respectively, implying that C3 and C4 behave as electrophiles.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Exploring the Potential Energy Surface (PES)\u003c/h2\u003e\u003cp\u003eDue to asymmetry of the reactants, the 32CA (1,3-dipolar cycloaddition) reaction among nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e can proceed through four competing reaction paths. These encompass two regioisomeric pathways: \u003cem\u003eortho\u003c/em\u003e and \u003cem\u003emeta\u003c/em\u003e, as well as two stereoisomeric orientations: \u003cem\u003eendo\u003c/em\u003e and \u003cem\u003eexo\u003c/em\u003e. As a result, the molecules (\u003cb\u003eTS1-ex\u003c/b\u003e, \u003cb\u003eTS1-en\u003c/b\u003e, \u003cb\u003eTS2-ex\u003c/b\u003e, with \u003cb\u003eTS2-en)\u003c/b\u003e have been identified, each leading to a corresponding cycloadduct: (\u003cb\u003eIC-3\u003c/b\u003e, \u003cb\u003eIC-4\u003c/b\u003e, \u003cb\u003eIC-5\u003c/b\u003e, with \u003cb\u003eIC-6\u003c/b\u003e) respectively. These TSs and products have been identified and described (see Scheme \u003cspan refid=\"Sch3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e summarizes the energetic also electronic properties of the 32CA stationary points reaction. Nonetheless, these values were computed in toluene using M06-2X-D3/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p) theoretical method. However, the table shows cycloadducts (\u003cb\u003eIC-3\u003c/b\u003e to \u003cb\u003eIC-6\u003c/b\u003e), TSs (\u003cb\u003eTS1-en\u003c/b\u003e, \u003cb\u003eTS1-ex\u003c/b\u003e, \u003cb\u003eTS2-en\u003c/b\u003e, \u003cb\u003eTS2-ex\u003c/b\u003e), the data on electronic energy (ΔE), enthalpy (ΔH), Gibbs free energy (ΔG), also entropy (ΔS). Among the cycloadducts, \u003cb\u003eIC-3\u003c/b\u003e is identified as the greatest thermodynamically stable, exhibiting the lowest ΔG of -8.92 kcal.mol\u003csup\u003e-1\u003c/sup\u003e, while \u003cb\u003eIC-5\u003c/b\u003e ranks as the least favorable with the maximum ΔG of -1.35 kcal.mol\u003csup\u003e-1\u003c/sup\u003e [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Among the TSs, \u003cb\u003eTS1-en\u003c/b\u003e displays the lowest ΔG, indicating it is the greatest kinetically favorable reaction path. It also has the highest GEDT value, suggesting a strong polar character and efficient electron transfer between the reactants. \u003cb\u003eTS1-ex\u003c/b\u003e is slightly higher in energy and has a comparable GEDT value, making it the second most favorable. In contrast, \u003cb\u003eTS2-en\u003c/b\u003e and \u003cb\u003eTS2-ex\u003c/b\u003e exhibit higher ΔG and lower GEDT values, indicating less favorable and less polar TSs.\u003c/p\u003e\u003cp\u003eTable 3 Using M06-2X-D3/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p), the 32CA reaction with GEDT stationary points were estimated in toluene at 110\u0026deg;C. The relative energies (∆E), enthalpies (∆H), Gibbs free energies (∆G), also entropies (∆S) kcal.mol\u003csup\u003e-1\u003c/sup\u003e were determined using average electron count.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStructure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eΔE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eΔH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eΔG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eΔS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGEDT\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIC-3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-23.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-24.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-8.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-51.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-------\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIC-4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-21.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-22.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-6.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-52.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-------\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIC-5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-16.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-17.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-53.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-------\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIC-6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-20.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-21.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-5.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-54.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-------\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS1-en\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-47.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0594 (FEDF)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS1-ex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-51.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0574 (FEDF)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS2-en\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-52.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0199 (NEDF)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS2-ex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-57.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0229 (NEDF)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe analysis of thermodynamic parameters reinforces that \u003cem\u003eortho\u003c/em\u003e\u003cb\u003e-TS1-en\u003c/b\u003e reaction path is the utmost favorable route, both thermodynamically and kinetically (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result supports experimental findings [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The GEDT values reflect the extent of electron transfer, with \u003cb\u003eTS1-en\u003c/b\u003e exhibiting the highest GEDT value (0.0594), indicating a tendency toward a polar reaction and classified as FEDF, suggesting a significant electron density flux. \u003cb\u003eTS2-en\u003c/b\u003e has a GEDT value of 0.0199, indicating a preference for a non-polar reaction and categorized as NEDF [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA representation of the optimized structures of the TSs via bond distances of O1\u0026ndash;C5 and C3\u0026ndash;C4 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, highlighting the asynchronicity in the 32CA reaction path. In the \u003cb\u003eTS1-en\u003c/b\u003e structure, the O1\u0026ndash;C5 bond distance is 2.16 \u0026Aring;, however the C3\u0026ndash;C4 bond is 2.08 \u0026Aring;, indicating a small change among the two forming bonds. However, in \u003cb\u003eTS1-ex\u003c/b\u003e, the O1\u0026ndash;C5 bond is 2.03 \u0026Aring; and C3\u0026ndash;C4 bond is 2.14 \u0026Aring;, showing a larger difference in bond lengths. The greater disparity in \u003cb\u003eTS1-ex\u003c/b\u003e suggests a more asynchronous bond formation compared to \u003cb\u003eTS1-en\u003c/b\u003e, where the bond formation is more synchronous. Therefore, \u003cb\u003eTS1-ex\u003c/b\u003e is more asynchronous than \u003cb\u003eTS1-en\u003c/b\u003e based on the bond distance differences [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. How synchronous the structure is (Δd, in \u0026Aring;) is measured by comparing lengths of the two forming sigma bonds. Moreover, in the case of \u003cb\u003eTS1-ex\u003c/b\u003e, a Δd of 0.11 \u0026Aring; between the C3\u0026ndash;C4 with O1\u0026ndash;C5 bonds indicates an asynchronous bond formation, though the bonds form nearly simultaneously. Additional Δd values for other TSs are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for comparison.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Analysis of the Topological Shape of ELF at TSs\u003c/h2\u003e\u003cp\u003eTopological analysis provides insights into electronic structures of optimal TSs participating in 32CA reaction between nitrone \u003cb\u003eGN-\u003c/b\u003e1 and \u003cb\u003eCA-2\u003c/b\u003e. The examination of their ELF analyzed the electrical structures of the TSs. Otherwise, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrates basin attractor positions with ELF localization zones for 32CA reactions' TSs. However, this analysis emphasizes bonding interactions among O1-C5 with C3-C4 in the \u003cem\u003eortho\u003c/em\u003e reaction path, as well as O1-C4 with C3-C5 in the \u003cem\u003emeta\u003c/em\u003e reaction path [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The ELF investigation of four reaction paths in 32CA reaction involving nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e reveals V(O1) also V\u0026prime;(O1) basins populations of 5.75, 5.67, 5.85, besides 5.78e for \u003cb\u003eTS1-ex\u003c/b\u003e, \u003cb\u003eTS1-en\u003c/b\u003e, \u003cb\u003eTS2-ex\u003c/b\u003e, with \u003cb\u003eTS2-en\u003c/b\u003e, corresponding to non-bonding electron density on O1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Admittedly, according to the ELF, in the region where N2-C3 bonds are formed, a V(N2,C3) disynaptic basin has been identified. Notably, the N2\u0026ndash;C3 bond region has 3.68 electrons in the nitrone \u003cb\u003eGN-1\u003c/b\u003e, but this decreases to 2.67, 2.57, 2.51, and 2.32 electrons in \u003cb\u003eTS2-en\u003c/b\u003e, \u003cb\u003eTS2-ex\u003c/b\u003e, \u003cb\u003eTS1-ex\u003c/b\u003e, and \u003cb\u003eTS1-en\u003c/b\u003e, respectively. Through this reduction, a \u003cem\u003epseudoradical\u003c/em\u003e center was produced at carbon atom C3, non-bonding electron density is produced at the nitrogen atom N2. Furthermore, ELF analysis also finds additional disynaptic basin, V(O1,N2), spanning 1.20-1.31e, indicating a single O1\u0026ndash;N2 bond in the nitrone \u003cb\u003eGN-1\u003c/b\u003e framework. In addition, each TS also features a disynaptic basin, V(C4,C5), which has a charge distribution of 2.78-2.70e, corresponds to the C4\u0026ndash;C5 bond in the \u003cb\u003eCA-2\u003c/b\u003e framework. However, \u003cb\u003eTS1-ex, TS1-en, TS2-ex\u003c/b\u003e, with \u003cb\u003eTS2-en\u003c/b\u003e indicate a monosynaptic basin V(C3) with values 0.79, 0.48, 0.27, and 0.34e, linked to the C3 carbon atom (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, \u003cb\u003eTS1-en\u003c/b\u003e, monosynaptic basins on C3 with C4 have populations of 0.48 with 0.33 e, respectively, indicating developing or partial lone pair character. The N2 lone pair shows a population of 1.21 electrons. Moreover, in \u003cb\u003eTS1-ex\u003c/b\u003e, the monosynaptic basins on C3 and C4 each possess values of 0.79 e, whereas N2 maintains 1.16 e, indicating a more asynchronous bonding process and greater lone pair contribution from the carbon atoms. In \u003cb\u003eTS2-en\u003c/b\u003e, C3 and C5 have lower monosynaptic populations of 0.34 and 0.35 electrons, and N2 has 0.88. \u003cb\u003eTS2-ex\u003c/b\u003e, the monosynaptic populations on C3 and C5 are 0.27 and 0.29 electrons, and N2 has 1.17 electrons, indicating slightly weaker bonding interactions and partial electron localization [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In contrast, all four competitive reaction paths are defined by the nonappearance of novel disynaptic basins linked to the anew established bonds C3\u0026ndash;C4/O1\u0026ndash;C5 and C3\u0026ndash;C5/O1\u0026ndash;C4 for the \u003cem\u003eortho\u003c/em\u003e with \u003cem\u003emeta\u003c/em\u003e reaction paths, respectively. However, this indicates the TSs have not yet formed two new covalent connections. The NCI analysis and QTAIM topological analysis study in Section \u003cspan refid=\"Sec11\" class=\"InternalRef\"\u003e3.7\u003c/span\u003e also supports this observation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e3.5 How the 32CA reaction works along the different reaction paths, as explained by the Bonding Evolution Theory (BET)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBET, developed by Krokoidis, predicts chemical reaction processes [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Within the 32CA reaction between nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e, the MEDT employed BET to investigate how electron density varies throughout the reaction path. This comprehensive analysis of the bonding pattern along the \u003cem\u003eortho\u003c/em\u003e-regioisomeric pathways identified six ELF topological phases. In general, Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Scheme \u003cspan refid=\"Sch3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, also Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e demonstrate the ELF attractor positions for six Structural Stability Domains (SSDs) (\u003cb\u003eSSD-1\u003c/b\u003e to \u003cb\u003eSSD-VI\u003c/b\u003e) determined along the IRC connected to the \u003cb\u003eTS1-en\u003c/b\u003e of the 32CA reaction path. Meanwhile, this method emphasizes O1-C5 and C3-C4 bonds. Certainly, \u003cb\u003eTS1-en\u003c/b\u003e reaction paths' electron density varies through bond creation and breaking, as seen by ELF attractors, resulting in the creation of the 32CA product. However, the step-by-step development and evolution of ELF attractors within structures \u003cb\u003eSSD-1\u003c/b\u003e through \u003cb\u003eSSD-VI\u003c/b\u003e indicate the process of bond creation and the stabilization of the product structure.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIn order to determine (\u003cb\u003eTS1-en\u003c/b\u003e) reaction pathway of the 32CA reaction, which incorporates nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e, ELF valence basin populations of the IRC structures \u003cb\u003eSSD-1\u003c/b\u003e to \u003cb\u003eSSD-VI\u003c/b\u003e are crucial.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eVI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003estructure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eSSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSSD (TS)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eSSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eSSD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed(O1-C5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed(C3-C4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGEDT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.073\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.252\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.307\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(O)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV'(O)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(O1,N2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(N2,C3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(C4,C5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(C3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(C4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(C3,C4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(N2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(C5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV(O1,C5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows that the electron density of the \u003cb\u003eTS1-en\u003c/b\u003e reaction path fluctuates during bond formation and cleavage, as shown by ELF attractors, which are important for understanding the reaction path of 32CA reaction involving nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e. Correspondingly, the table monitors changes in bond distances (such as d(O1-C5), d(C3-C4)), GEDT, and ELF populations throughout different stages of the reaction. Importantly, as the reaction moves forward, bond distances decrease (for instance, O1-C5 declines from 3.04 to 1.44), indicating the formation of bonds. The GEDT values increase, suggesting enhanced electron transfer. ELF populations (like V(N2,C3), V(C4,C5)) demonstrate shifts in electron density, with certain basins emerging or disappearing, illustrating the reorganization of electrons during the reaction [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the progression of bond creation beside the reaction path of \u003cb\u003eTS1-en\u003c/b\u003e in the 32CA reaction among nitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e. However, the figure highlights the particular points along the IRC for each SSD structure, emphasizing the evolution of key interactions during the reaction. The depiction illustrates the involvement of atoms O1, C3, C4, N2, and C5 at various stages of the reaction, with specific valence basins corresponding to different phases. The appearance and disappearance of these valence basins indicate significant electronic rearrangements. The presence of V(C3) in the early structures suggests an initial localisation of electron density at C3, which later transforms as the reaction progresses. The emergence of V(C3,C4) also V(O1,C5) in later stages indicates the making of new C\u0026ndash;C with O\u0026ndash;C bonds. Whenever V(C4) and V(N2) also appear at specific stages, contributing to the reaction mechanism. The red ellipse in the figure highlights the regions where topological alterations occur, marking critical points of electronic reorganization. These changes confirm the gradual bond creation process alongside the reaction coordinate, supporting the transition from reactants to products. The figure effectively captures the evolution of electron density and bonding interactions, providing a visual representation of the reaction mechanism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSix distinct topological phases along the \u003cb\u003eTS1-en\u003c/b\u003e chemical pathway were found in ELF basins. The initial \u003cb\u003eSSD-I\u003c/b\u003e ELF structure mirrors the bonding forms in the compounds. Moreover, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, ELF analysis at \u003cb\u003eTS1-en\u003c/b\u003e indicates a V(N1) monosynaptic basin, containing approximately 1.1 electrons, at \u003cb\u003eSSD-II\u003c/b\u003e, which corresponds to the lone pair on N2 nitrogen. In addition, \u003cem\u003epseudoradical\u003c/em\u003e centers are also found at carbon atom C3 within same \u003cb\u003eSSD-II\u003c/b\u003e, thus leading to the development of monosynaptic basin V(C3). However, \u003cem\u003epseudoradical\u003c/em\u003e centers identified at C4 carbon atoms in \u003cb\u003eSSD-III\u003c/b\u003e, the monosynaptic basin V(C4) significant contributor to its composition. In general, \u003cb\u003eTS1-en\u003c/b\u003e is part of \u003cb\u003eSSD-III\u003c/b\u003e, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. However, the O1\u0026ndash;C5 with C3\u0026ndash;C4 single bonds have not yet been established. Frequently, in \u003cb\u003eSSD-IV\u003c/b\u003e, first single bonds occur among C3 with C4.\u003c/p\u003e\u003cp\u003eAdditionally, in this phase, \u003cb\u003eSSD-IV's\u003c/b\u003e C5 carbon atoms have pseudoradical centers, creating the monosynaptic basin V(C5). A new bond is formed between O1 and C5 in \u003cb\u003eSSD-V\u003c/b\u003e [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eScheme \u003cspan refid=\"Sch3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the key electronic and structural changes during the 32CA reaction among nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e, focusing on the \u003cb\u003eTS1-en\u003c/b\u003e reaction path. It highlights sequential bonding modifications, such as the shortening of bond distances (e.g., V(C4,C5) decreases from 3.26 to 2.01) and shifts in electron density (e.g., V(N2,C3) drops from 3.68 to 1.8), indicating bond formation and reorganization. The GEDT values increase as the reaction progresses, reflecting enhanced electron transfer. New basins like V(C3,C4) with V(O1,C5) emerge in later stages, signifying the creation of new bonds. The \u003cb\u003eTS\u003c/b\u003e (\u003cb\u003eSSD-III\u003c/b\u003e) is marked by intermediate values, confirming its role as the critical point in the reaction. Overall, the scheme captures the dynamic evolution of electron populations and bonding interactions along the reaction path [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.6 NCI analysis\u003c/h2\u003e\u003cp\u003eNCI are crucial in influencing the stability with reactivity of molecular systems [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The NCI analysis was performed on the TS structures (\u003cb\u003eTS1-ex\u003c/b\u003e, \u003cb\u003eTS1-en\u003c/b\u003e, \u003cb\u003eTS2-ex\u003c/b\u003e, with \u003cb\u003eTS2-en\u003c/b\u003e) to verify existence with significance of these interactions in stabilizing the TSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). NCI gradient isosurfaces reveal weak intermolecular forces for example Van der Waals interactions, hydrogen bonds, aslo steric effects repulsions. Significantly, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e demonstrations the four TS scatter plots. However, the RDG for λ2\u0026thinsp;\u0026gt;\u0026thinsp;0 and λ2\u0026thinsp;\u0026lt;\u0026thinsp;0 identifies dominating interactions based on (λ2)ρ sign. Therefore, the scatter plot of \u003cb\u003eTS1-en\u003c/b\u003e and \u003cb\u003eTS2-ex\u003c/b\u003e shows a blue region (-0.05 to -0.02 a.u.), thin green band, with a red zone. Usually, for the other TSs, the blue region gets smaller, and the red region becomes a bit smaller as the green region expands [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. A significant attraction (blue) exists among O1 with C3 atoms of nitrone \u003cb\u003eGN-1\u003c/b\u003e also C-C atoms (double bonds) of \u003cb\u003eCA-2\u003c/b\u003e across TSs, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.7 QTAIM topological analysis\u003c/h2\u003e\u003cp\u003eA QTAIM topological analysis of electron density ρ at critical points (CPs) in the molecular region involved in forming new C-C and O-C single bonds at TSs was conducted to understand better atomic interactions during the 32CA reaction between nitrone \u003cb\u003eGN-1\u003c/b\u003e and \u003cb\u003eCA-2\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The QTAIM variables are in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The newly selected critical points, CP1 and CP2, have low electron density ρ(r) values (below 0.1 a.u.) at the four TSs. The small positive \u0026nabla;\u0026sup2;ρr indicates no covalent bonding in these locations. Therefore, creation of new C\u0026ndash;C with O\u0026ndash;C bonds hasn't started at the TS, matching ELF study results. (see Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). For O1-C5, the electron density is highest in \u003cb\u003eTS1-ex\u003c/b\u003e (0.0693 a.u) and lowest in \u003cb\u003eTS1-en\u003c/b\u003e (0.0591 a.u), while the Laplacian values follow a similar trend. For C3-C4, \u003cb\u003eTS1-en\u003c/b\u003e shows the highest electron density (0.0751 a.u), with \u003cb\u003eTS2-ex\u003c/b\u003e has lowest (0.0595 a.u). The Laplacian for C3-C5 is highest in \u003cb\u003eTS2-ex\u003c/b\u003e (0.0405 a.u) and lowest in \u003cb\u003eTS1-en\u003c/b\u003e (0.0209 a.u). These values indicate variations in bond strength and character across the TSs [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. This analysis further highlights the asynchronicity shown by the distance progress indexes discussed in Section 3.5.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003enitrone \u003cb\u003eGN-1\u003c/b\u003e with \u003cb\u003eCA-2\u003c/b\u003e undergo a 32CA reaction. This reaction is affected by the entire electron density ρ (a.u.) also Laplacian of the electron density \u0026nabla;\u0026sup2;ρ(rc), (a.u.), at critical points in the TSs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTSs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eCP1(O1-C5\u003csup\u003ea\u003c/sup\u003e/C4\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eCP2 (C3-C4\u003csup\u003ea\u003c/sup\u003e/C5\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e\u0026#120646;\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026#120513;\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026#120784;\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e\u0026#120646;\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026#120646;\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026#120513;\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026#120784;\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e\u0026#120646;\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS1-en\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0591\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.119\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0751\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0209\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS1-ex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0301\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS2-en\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0644\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.124\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0610\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0350\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTS2-ex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0634\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0595\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.0405\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e for \u003cb\u003eortho\u003c/b\u003e and \u003csup\u003eb\u003c/sup\u003e for \u003cb\u003emeta\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Molecular docking study against PDB ID: 2ITZ\u003c/h2\u003e\u003cp\u003eMolecular docking simulations predict the optimal way a ligand binds to a macromolecular target. They create numerous postures at protein's binding position. Furthermore, best ligand conformations were chosen depending on optimum organization and the lowest free binding energy [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Additionally, Flare 9 was utilized for molecular docking investigations, and conventional methods and methodology were adhered to. Moreover, using this method, molecules \u003cb\u003eIC-3\u003c/b\u003e, \u003cb\u003eIC-4\u003c/b\u003e, \u003cb\u003eIC-5\u003c/b\u003e, with \u003cb\u003eIC-6\u003c/b\u003e were attached to the active site of the EGFR L858R mutation, in combination with Gefitinib (PDB ID: 2ITZ). This entry showcases the EGFR kinase domain harboring the L858R mutation, a common alteration in lung cancer, in complex with the drug gefitinib (Iressa). Such mutations can affect drug binding and influence treatment efficacy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e illustrates the comparison between the original and re-docked poses of a ligand-protein complex\u003c/p\u003e\u003cp\u003eThe RMSD value of 1.439 \u0026Aring; (angstroms) measures the difference between the two positions. RMSD (Root Mean Square Deviation) is a common metric used to assess how accurately a docking method can replicate the experimentally established position of a ligand [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e presents the binding affinities of four molecules \u003cb\u003eIC-3, IC-4, IC-5\u003c/b\u003e, and \u003cb\u003eIC-6\u003c/b\u003e along with the co-crystal ligand, measured in kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e against the 2ITZ target. However, amongst the established compounds, \u003cb\u003eIC-6\u003c/b\u003e shows the highest binding affinity at -10.19 kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, followed by \u003cb\u003eIC-4\u003c/b\u003e and \u003cb\u003eIC-5\u003c/b\u003e. \u003cb\u003eIC-3\u003c/b\u003e has the weakest binding among the new compounds. The co-crystal ligand demonstrates the highest binding affinity overall at -11.43 kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, serving as a benchmark for comparison. These results suggest that \u003cb\u003eIC-6\u003c/b\u003e is the most promising candidate among the tested compounds [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ekcal.mol\u003csup\u003e-1\u003c/sup\u003e values indicate the binding affinity of compounds \u003cb\u003eIC-3\u003c/b\u003e, \u003cb\u003eIC-4, IC5\u003c/b\u003e, and \u003cb\u003eIC-6\u003c/b\u003e to the 2ITZ, as well as the co-crystal ligands.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAffinity (kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIC-3\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIC-4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIC-5\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIC-6\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCo-crystal ligand\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-8.33\u003c/p\u003e\u003cp\u003e-9.63\u003c/p\u003e\u003cp\u003e-9.23\u003c/p\u003e\u003cp\u003e-10.19\u003c/p\u003e\u003cp\u003e-11.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFigure 12 illustrates the interaction of molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5,\u003c/strong\u003e with \u003cstrong\u003eIC-6\u003c/strong\u003e with the EGFR L858R mutant (PDB ID: 2ITZ) using both 3D and 2D representations. The 3D visualization offers a spatial perspective on how these compounds fit within the binding site, displaying molecular conformations and potential interactions such as hydrogen bonding, hydrophobic interactions, or electrostatic forces [87, 88]. The 2D representation highlights specific molecular interactions among the compounds also amino acid residues of target, simplifying the analysis of binding mechanisms. This combined visualization approach facilitates an understanding of how effectively these compounds bind to and interact with the target protein. Table S2 provides a summary of the binding affinities and nonbonding interactions among the 2ITZ target, molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e, \u003cstrong\u003eIC-6\u003c/strong\u003e, with co-crystallized ligand.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.9 Evaluation of drug-likeness and ADMET predictions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAn ADMET revision assesses a drug\u0026apos;s pharmacokinetics by analyzing Absorption, Distribution, Metabolism, Excretion, with Toxicity. Meanwhile, it predicts how a drug behaves also its effects in the body, especially regarding oral also intestinal absorption, which is crucial in drug detection. However, unfortunate absorption can hinder distribution and metabolism, principal to risks like neurotoxicity and nephrotoxicity. Additionally, Table 7 shows the SwissADME-predicted drug-likeness properties for molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e, with \u003cstrong\u003eIC-6\u003c/strong\u003e. All four compounds have the same molecular weight (369.16 g/mol), fall within the optimal range, and share identical values for number of hydrogen donors (nHD) (1), hydrogen bond acceptors (nHA) (6), Number of rotatable bonds (nROT) (8), and topological polar surface area (TPSA) (76.07 \u0026Aring;\u0026sup2;). Their LogP values, indicating lipophilicity, are also within the favorable range, with \u003cstrong\u003eIC-4\u003c/strong\u003e being the most lipophilic. All compounds fully adhere to Lipinski\u0026rsquo;s Rule of Five, indicating favorable potential for oral bioavailability. These results indicate that each compound possesses favorable drug-like properties [78]. Hence, these findings demonstrate that all three substances possess features that are similar to those of drugs, making them strong candidates for additional progress in drug finding. Their dependable adherence to Lipinski\u0026apos;s rules is especially promising for their potential as drug applicants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7.\u0026nbsp;\u003c/strong\u003eSwissADME calculated drug-likeness for \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e, and \u003cstrong\u003eIC-6\u003c/strong\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProperty\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC-3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC-4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC-5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC-6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eMW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e369.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e369.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e369.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e369.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eContain hydrogen atoms. Optimal:100~600\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003enHD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eNumber of hydrogen bond donors. Optimal:0~7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003enROT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eNumber of rotatable bonds. Optimal:0~11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eLogP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2.223\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1.766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eThe logarithm of the n-octanol/water distribution\u003c/p\u003e\n \u003cp\u003ecoefficients at pH=7.4.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003enHA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eNumber of hydrogen bond acceptors. Optimal:0~12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eTPSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e76.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e76.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e76.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e76.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eTopological Polar Surface Area. Optimal:0~140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eLipinski rule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e(Accepted)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e(Accepted)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e(Accepted)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e(Accepted)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cul\u003e\n \u003cli\u003eMW \u0026nbsp; 500; logP \u0026nbsp; 5; Hacc \u0026nbsp; 10; Hdon \u0026nbsp; 5 n\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIf two properties are out of range, a poor absorption or permeability is possible; one is acceptable.\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e:\u0026nbsp;MW, Molecular weight;\u0026nbsp;nHD, number of hydrogen donors;\u0026nbsp;nROT, Number of rotatable bonds; LogP, Log of the octanol/water partition coefficient; nHA, number of hydrogen acceptors; TPSA, topological polar surface area;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The boiled egg model, shown in Figure 13, estimates chemical component absorption in the gastrointestinal system with blood-brain barrier crossing based on WLOGP against TPSA reference plot locations. However, this illustration shows that the yolk implies brain penetration and white region suggests strong passive absorption in gastrointestinal system. The positioning of the compounds within these regions determines their pharmacokinetic behavior. If a compound is located within the yolk, it suggests a high likelihood of crossing the blood-brain barrier, thereby increasing its potential to reach central nervous system. If it is located in the white region, it is more likely to be well absorbed in the intestinal tract, but not necessarily to penetrate the brain. The presence of \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e with \u003cstrong\u003eIC-6\u003c/strong\u003e within yolk indicates that these compounds are expected to cross blood-brain barrier (BBB) successfully. Particularly, this prediction is significant for calculating the potential activity of the compounds within the central nervous system [89, 90].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 14 presents a physicochemical radar chart that visually represents the key molecular properties of the selected compounds. This chart provides an overview of multiple parameters simultaneously, offering insights into the balance of different physicochemical characteristics. Each axis of the radar chart corresponds to a specific property, such as lipophilicity, polarity, solubility, molecular weight, flexibility, and saturation. Table S3 summarizes the SwissADME-predicted distribution and excretion properties of molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e, with \u003cstrong\u003eIC-6\u003c/strong\u003e. As shown in Table S3, the BBB permeability values for molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e with \u003cstrong\u003eIC-6\u0026nbsp;\u003c/strong\u003eare 0.084, 0.206, 0.066, and 0.396, respectively, indicating that compound \u003cstrong\u003eIC-6\u003c/strong\u003e has the highest potential to cross the blood-brain barrier. However, \u003cstrong\u003eIC-5\u003c/strong\u003e shows the lowest. Regarding human liver microsomal (HLM) stability, \u003cstrong\u003eIC-4\u003c/strong\u003e and \u003cstrong\u003eIC-5\u003c/strong\u003e are likely to be less stable, with values closer to 1 indicating a higher probability of metabolic instability. For plasma clearance (CL), all compounds fall within the moderate clearance range (5\u0026ndash;15 ml/min/kg), with \u003cstrong\u003eIC-6\u003c/strong\u003e having the highest predicted clearance. These results provide insight into the compounds\u0026rsquo; pharmacokinetic behaviors[74]. Table S4 shows SwissADME-based drug-likeness predictions for four molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5\u003c/strong\u003e, with \u003cstrong\u003eIC-6\u003c/strong\u003e, emphasizing their relations with cytochrome-P (CYP) enzymes. However, Table S5 shows the ADMET/pharmacokinetic properties of the best-hit compounds, focusing on key metrics such as permeability, Pgp interactions, and bioavailability. Table S6 summarizes the predicted toxicity profiles of molecules \u003cstrong\u003eIC-3\u003c/strong\u003e, \u003cstrong\u003eIC-4\u003c/strong\u003e, \u003cstrong\u003eIC-5,\u003c/strong\u003e with \u003cstrong\u003eIC-6\u003c/strong\u003e, evaluated utilizing OSIRIS Property Explorer and PreADMET.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.10 Evaluation of antibacterial activity utilizing PASS for the examined substances\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial activity of compound \u003cstrong\u003eIC-3\u003c/strong\u003e was assessed using PASS \u003cspan dir=\"RTL\"\u003e[91]\u003c/span\u003e. Moreover, the PASS tool provides a simple and efficient method for estimating a compound\u0026apos;s potential biological activities [92, 93]. Additionally, it facilitates the early assessment of a compound\u0026apos;s pharmacological profile, encompassing therapeutic benefits, mechanisms of action, and safety aspects, including toxicity and side effects. However, the findings are encapsulated in Tables 8 and S7. Moreover, the PASS prediction outcomes show that \u003cstrong\u003eIC-3\u003c/strong\u003e has strong pharmacological potential, as all compounds show activity probabilities (Pa) above 0.7. However, Table 8 shows that \u003cstrong\u003eIC-3\u003c/strong\u003e has a great probability of acting as an aldose reductase substrate, with a probability of activity (Pa) value of 0.842. The probability of inactivity (Pi) is very low at 0.002. This indicates strong confidence that \u003cstrong\u003eIC-3\u003c/strong\u003e may interact with or be metabolised by aldose reductase, suggesting potential relevance in biological or pharmacological contexts involving this enzyme [94, 95]. Table S7 lists the predicted biological activities of the \u003cstrong\u003eIC-3\u003c/strong\u003e compound, showing multiple potential enzyme interactions and pharmacological effects based on PASS analysis with a threshold of Pa \u0026gt; 0.3. The high probabilities for targets such as CDP-glycerol glycerophosphotransferase and various dehydrogenases highlight \u003cstrong\u003eIC-3\u003c/strong\u003e\u0026rsquo;s potential multifunctional bioactivity, which may be valuable in drug discovery or biochemical\u0026nbsp;reaction path\u0026nbsp;modulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8\u003c/strong\u003e. PASS prediction of \u003cstrong\u003eIC-3\u003c/strong\u003e compound\u0026apos;s main potential activities. The results indicate the probability of a molecule being active, with Pa \u0026gt; 0.7.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"391\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 290px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiological Activity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAldose reductase substrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePa = Probability to be active, Pi = Probability to be inactive.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.11 Molecular Dynamics stimulation\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe compound \u003cstrong\u003eIC-6\u003c/strong\u003e which showed the best binding affinity against the target proteins (EGFR kinase domain harboring the L858R mutation), \u0026nbsp;were further considered for MD simulation study using Gromacs-2025 software on a GPU system [52]. The protein-ligand complexes generated from the binding affinity prediction analysis were subjected to MD simulation study along with the original crystal structure of the target protein complexed with the co-crystal inhibitors. The protein-ligand complexes were initially cleaned and prepared using UCSF Chimera 1.18. The energy minimization process was performed using the steepest descent method, and Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e ions at the concentration of 0.1M were added for neutralization. All essential topology files, including CHARMM parameter files, were generated using the SwissParam server [96]. MD simulations of the complex were conducted for a total simulation time of 100 nanoseconds (ns), collecting snapshots at intervals of 100 picoseconds (ps) from 0 ns to 100 ns at 1 bar and 300 K reference pressure and temperature. The root mean square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) were analyzed for the target proteins (EGFR kinase domain harboring the L858R mutation) and complex forms to test the conformational stability. Figure 15 presents three important MD analysis plots for the nonstructural protein\u0026ndash;ligand complex (\u003cstrong\u003eIC-6-2ITZ\u003c/strong\u003e): RMSD, RMSF, and Radius of Gyration (Rg), each offering insight into the structural stability, flexibility, and compactness of the complex during the simulation period. The root mean square deviation (RMSD) plot (A) illustrates the root mean square deviation of the protein\u0026ndash;ligand complex backbone atoms over time, reflecting the structural stability of the complex. A low and relatively constant RMSD curve suggests that the complex remains stable throughout the simulation, with no major conformational changes. If the RMSD increases sharply or fluctuates heavily, this would indicate instability or significant structural rearrangements. The structural changes of the \u003cstrong\u003eIC-6-2ITZ\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/strong\u003ecomplex were examined using RMSD. The conformational change of Ligand atoms was estimated and compared to the initial conformation over 100 ns of MD simulations. As can be seen in Figure 15 (A), the overall stability of the investigated complex was observed with a protein\u0026rsquo;s RMSD value ranging from 0.3 to 1.0 nm. Its fluctuations towards the end of the simulation are around some thermal average structure. From about 60 ns to the end of the simulation time, the RMSD values of the protein stabilize at around 0.75 nm, indicating that the simulation converges, then the system has reached an equilibrium state. Furthermore, Backbone RMSD (red color) indicates how stable the Backbone is with respect to the protein and its binding pocket. At the end of the simulation time, the RMSD value of the Backbone is 0.25 nm, lower than that of ligand by 0.5. Therefore, it is certain that the ligand has not diffused away from its initial binding site. Overall, these results confirmed that the \u003cstrong\u003eIC-6\u003c/strong\u003e inhibitor is tightly bonded and does not affect the overall topology of EGFR. The RMSF plot (B) shows the root mean square fluctuation of each residue, indicating the flexibility of individual amino acids. Higher RMSF values correspond to regions with greater flexibility, such as loops or terminal ends, while lower values are typical of more rigid, structured regions like \u0026alpha;-helices or \u0026beta;-sheets. This analysis helps identify which parts of the protein are most dynamic or possibly involved in ligand interactions. The RMSF value for the same complex is mainly below \u003cspan dir=\"RTL\"\u003e1\u003c/span\u003e.\u003cspan dir=\"RTL\"\u003e3\u003c/span\u003e nm with the strongest fluctuations observed for the residue positions \u003cspan dir=\"RTL\"\u003e700\u003c/span\u003e, \u003cspan dir=\"RTL\"\u003e722\u003c/span\u003e and \u003cspan dir=\"RTL\"\u003e1007\u003c/span\u003e Figure 15 (B). The Radius of gyration (Rg) plot (C) measures the radius of gyration, which provides information about the compactness of the protein structure over time. A consistent Rg value throughout the simulation suggests that the protein maintains its structural integrity and does not undergo significant expansion or contraction. On the other hand, notable changes in the Rg value may indicate partial unfolding or a change in compactness due to ligand binding or conformational drift. Rg values for this complex form a relatively stable profile from 0.37 nm to 0.435 nm Figure 15 (C).\u0026nbsp;\u003c/p\u003e"},{"header":"4.\tConclusions","content":"\u003cp\u003eThe study provided deep mechanistic insights into 32CA reaction among glutaraldehyde-N-aryl nitrone with cinnamaldehyde using MEDT. However, the results indicated the reaction adheres to a two-stage, one-step mechanism, aligning with study of bond formation developments. Moreover, global reactivity indices indicated a polar property of process, while nitrone tends to be a strong nucleophile, and cinnamaldehyde is an electrophile. ELF and topological analysis confirmed that the reaction occurs through non-concerted bond formation, validating the stepwise character of the mechanism. Furthermore, GEDT analysis revealed that the reaction pathways are highly asynchronous, showing differences in electron density transfer along regioisomeric channels. Energetic profiles established that the \u003cem\u003eortho\u003c/em\u003e and \u003cem\u003eendo\u003c/em\u003e pathways are energetically favored, which aligns with the observed regioselectivity and stereoselectivity. Otherwise, the analysis too confirmed polar nature process plays a decisive role in controlling selectivity, in addition to the mechanistic study, molecular docking, ADMET, with MD simulations used to investigate the biological potential of the resulting cycloadducts. Docking studies suggested strong binding affinities of the products toward biological targets, with favorable pharmacokinetic profiles predicted by ADMET analysis. MD confirmed the stability of ligand\u0026ndash;receptor complexes, reinforcing the potential of these molecules as bioactive agents. Overall, the study not only clarified the mechanistic features of the [3+2] cycloaddition but also highlighted the biological relevance and drug-like properties of the synthesized compounds, making them promising candidates for further pharmacological development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e RNS,\u0026nbsp;HMS,\u0026nbsp;and MA calculated and planned the technique. HMS and RNS wrote the manuscript. The authors collectively conducted the discussion and manuscript revision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare that they did not receive any awards, cash, or other financial support for writing this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The writers confirm they have no conflicts of attention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e The authors express their gratitude to the University of Valencia for given that the essential computing resources for this investigation. However, all calculations presented in this paper were conducted using the Gaussian 16 software on a supercomputer. Additionally, we definite our thankfulness to Cresset for allowing us access to the academic version of Flare V9 Software, located at New Cambridge House, Litlington, Cambridgeshire, UK, for the purpose of conducting molecular docking experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e There was no generation or analysis of any datasets as part of the current investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u003c/strong\u003e not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePadwa A, Pearson W. Synthesis Applications of 1, 3-Dipolar Cycloaddition Chemistry: Wiley, New York; 1984.\u003c/li\u003e\n\u003cli\u003eFeuer H. Nitrile oxides, nitrones and nitronates in organic synthesis: novel strategies in synthesis: John Wiley \u0026amp; Sons; 2008.\u003c/li\u003e\n\u003cli\u003eAcharjee N, Mohammad-Salim HA, Chakraborty M. 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Journal of computational chemistry. 2011;32(11):2359-68.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"structural-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"stuc","sideBox":"Learn more about [Structural Chemistry](https://www.springer.com/journal/11224)","snPcode":"11224","submissionUrl":"https://submission.nature.com/new-submission/11224/3","title":"Structural Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"GEDT, FEDF, Molecular Docking, ADMET, Molecular Dynamics","lastPublishedDoi":"10.21203/rs.3.rs-7773346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7773346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis research provides a thorough computational analysis of the [3\u0026thinsp;+\u0026thinsp;2] cycloaddition (32CA) reaction involving Glutaraldehyde-N-aryl nitrone (\u003cb\u003eGN-1\u003c/b\u003e) with Cinnamaldehyde (\u003cb\u003eCA-2\u003c/b\u003e), utilizing Molecular Electron Density Theory (MEDT) as the interpretive framework. However, the potential energy surface analysis identified four stereoisomeric pathways, with the \u003cem\u003eortho\u003c/em\u003e-\u003cem\u003eendo\u003c/em\u003e channel becoming thermodynamically and kinetically better. However, Global Electron Density Transfer (GEDT) values at the transition states (TSs) showed a clear forward electron density flux (FEDF), confirming the polar nature of the main mechanism. Electron Localization Function (ELF) with Bonding Evolution Theory (BET) analyses highlighted asynchronous bond formation along the preferred pathway. The stability of the cycloadducts was further assessed through molecular docking against the EGFR L858R mutant (PDB ID: 2ITZ), where compound \u003cb\u003eIC-6\u003c/b\u003e exhibited the strongest binding affinity. Molecular dynamics (MD) simulations of the \u003cb\u003eIC-6\u003c/b\u003e/EGFR complex over 100 ns validated its stable interaction profile and dynamic conformational behavior within the binding site. Furthermore, Absorption, Distribution, Metabolism, Excretion, with Toxicity (ADMET) predictions confirmed favorable drug-likeness with pharmacokinetic characteristics for synthesized compounds. Overall, integration of GEDT, FEDF, and MD provides deep mechanistic insights into the reaction pathway, while highlighting the therapeutic potential of the resulting isoxazolidine derivatives.\u003c/p\u003e","manuscriptTitle":"Mechanistic Insights into (3+2) Cycloaddition of Glutaraldehyde-N-Aryl Nitrone with Cinnamaldehyde: Electron Density, Docking, and Molecular Dynamics Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 11:31:55","doi":"10.21203/rs.3.rs-7773346/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-25T02:08:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T14:48:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T10:31:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209226656836240385576884339894476472380","date":"2025-10-13T12:13:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57391564063124818354152895217237537917","date":"2025-10-12T21:45:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-12T21:06:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-09T18:29:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-08T10:23:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Structural Chemistry","date":"2025-10-03T11:59:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"structural-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"stuc","sideBox":"Learn more about [Structural Chemistry](https://www.springer.com/journal/11224)","snPcode":"11224","submissionUrl":"https://submission.nature.com/new-submission/11224/3","title":"Structural Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dee7a732-7c62-41d5-8c93-c627b6628f35","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:45:57+00:00","versionOfRecord":{"articleIdentity":"rs-7773346","link":"https://doi.org/10.1007/s11224-025-02698-x","journal":{"identity":"structural-chemistry","isVorOnly":false,"title":"Structural Chemistry"},"publishedOn":"2026-01-16 16:28:31","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-10-27 11:31:55","video":"","vorDoi":"10.1007/s11224-025-02698-x","vorDoiUrl":"https://doi.org/10.1007/s11224-025-02698-x","workflowStages":[]},"version":"v1","identity":"rs-7773346","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7773346","identity":"rs-7773346","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}