Evaluation of Antimicrobial, Anticholinesterase Potential of Indole Derivatives and Unexpectedly Synthesized Novel Benzodiazine: Characterization, DFT and Hirshfeld Charge Analysis

The pharmacological effectiveness of indoles, benzoxazepines and benzodiazepines initiated our synthesis of indole fused benoxazepine/benzodiazepine heterocycles, along with enhanced biological usefulness of the fused rings. Activated indoles 5, 6 and 7 were synthesized using modified Bischler indole synthesis rearrangement. Indole 5 was substituted with the trichloroacetyl group at the C7 position, yielding 8, exclusively due to the increased nucleophilic character of C7. When trichloroacylated indole 8 was treated with basified ethanol or excess amminia, indole acid 9 and amide 10 were yielded, respectively. Indole amide 10 was expected to give indole fused benoxazepine/benzodiazepine 11a/11b on treatment with alpha halo ester followed by a coupling agent, but when the reaction was tried, an unexpectedly rearranged novel product, 1,3-bezodiazine 12, was obtained. The synthetic compounds were screened for anticholinesterase and antibacterial potential; results showed all products to be very important candidates for both activities, and their potential can be explored further. In addition, 1,3-bezodiazine 12 was explored by DFT studies, Hirshfeld surface charge analysis and structural insight to obrain a good picture of the structure and reactivity of the products for the design of derivatised drugs from the novel compound.

Recently, we have reported that some indoles with anti-cholinesterase activity are comparable to standard drugs. These compounds can be explored as a good alternative in AD therapeutics [8]. All synthesized indoles and 1,3-diazine were screened for their anticholinesterase potential. There is a continuous need to develop new antibacterial drugs to address the growing problem of emerging pathogenic infections and antibiotic resistance. Given the ability of indoles to combat bacterial infections, various types of indole derivatives have been explored for their potential antibacterial activities. Based on indoles' capability to fight against bacteria, natural indole alkaloids [25], bis-indoles [26], indole-containing hybrid [27], spirocyclic [28], and many other indole derivatives are being used as potential antibacterial agents. Regarding the significant antibacterial potential of indoles, synthesized rings are tested for antibacterial potential.
Considering this significance, the current study aims to conduct Hirshfeld surface analysis and Density Functional Theory (DFT) studies of the novel indole-fused benzodiazine compound (12). By conducting in-depth analyses (of specific characteristics and chemical reactivity) of this novel compound (indole-fused benzodiazine), we can better understand its potential as an effective pharmaceutical candidate and ultimately contribute to developing novel treatments for various diseases.

Synthesis
Owing to its pharmacological potential, it was planned to synthesize indole-based benzodiazepine/benzoxazepine. The retrosynthesis for the target compound is given in Scheme 1. We synthesized indoles (5-7) following our already-reported procedure [29]. The indoles (5-7) (Figures S1-S3) are activated and make only the C 7 highly electron-rich position, which acts as a good nucleophile and can undergo aromatic substitution reactions. The indole 5 was reacted with Cl 3 CCOCl in CH 2 Cl 2 to afford trichloroacetyl substituted indole 8 ( Figure S4). When indole 8 in CH 2 Cl 2 was stirred with aq. KOH or liquid NH 3 , this resulted in indole acid 9 ( Figure S5) and indole amide 10 ( Figure S6), respectively [30]. We attempted to convert the indole amide 10 into indole-fused benzodiazepine-1,3-dione (11a-b) by reacting with a coupling agent methyl 2-bromomethyl propionate (Scheme 2).
Molecules 2023, 28, x FOR PEER REVIEW 3 of 23 We synthesized indoles (5-7) following our already-reported procedure [29]. The indoles (5-7) (Figures S1-S3) are activated and make only the C 7 highly electron-rich position, which acts as a good nucleophile and can undergo aromatic substitution reactions. The indole 5 was reacted with Cl3CCOCl in CH2Cl2 to afford trichloroacetyl substituted indole 8 ( Figure S4). When indole 8 in CH2Cl2 was stirred with aq. KOH or liquid NH3, this resulted in indole acid 9 ( Figure S5) and indole amide 10 ( Figure S6), respectively [30]. We attempted to convert the indole amide 10 into indole-fused benzodiazepine-1,3-dione (11a-b) by reacting with a coupling agent methyl 2bromomethyl propionate (Scheme 2). When 5 was refluxed with Cl3CCOCl in CH2Cl2, the proton with indolic C 7 of 5 was substituted by a trichloro-acetyl group. The 1 H NMR of 8 showed the disappearance of H 7 ; the C 7 shifted downfield compared to the reactant, and a new carbonyl CO (C 8 ) signal emerged in the 13 C NMR spectrum of 8. These changes indicated the conversion of 5 to 8. Further confirmation was achieved by ESI MS of the 8 exhibiting molecular ion [M + H] +• at 474.0432 amu (found for C24H19 35 Cl3NO3) and the XRD structure was also proof of COCCl3 substitution (Figure 1) (File S1). Scheme 2. Reagents and conditions: (a) PhNH 2 , AcOH or NaHCO 3 (LiBr) reflux in EtOH; (b) Cl 3 CCOCl , CH 2 Cl 2 , reflux; (c) aq KOH in EtOH; (d) aq NH 3 , reflux; (e) NaH in DMSO (solid arrow), MeCH(Br)CO 2 Me (arrow with dashed lines).
When 5 was refluxed with Cl 3 CCOCl in CH 2 Cl 2 , the proton with indolic C 7 of 5 was substituted by a trichloro-acetyl group. The 1 H NMR of 8 showed the disappearance of H 7 ; the C 7 shifted downfield compared to the reactant, and a new carbonyl CO (C 8 ) signal emerged in the 13 C NMR spectrum of 8. These changes indicated the conversion of 5 to 8. Further confirmation was achieved by ESI MS of the 8 exhibiting molecular ion [M + H] +• at 474.0432 amu (found for C 24 H 19 35 Cl 3 NO 3 ) and the XRD structure was also proof of COCCl 3 substitution ( Figure 1) (File S1).
The trichlor-acetyl group, being a good leaving group, was conveniently substituted by OH or NH 3 to produce indole acid 9 or amide 10, accordingly, by refluxing indole 8 with basified EtOH or excess aqueous NH 3 in THF. The amide formation was authenticated by the appearance of two amide protons in the 1 H NMR. One proton emerged as a doublet (J = 3.0 Hz) at 7.81 ppm, and the other proton appeared as a singlet at 7.54 ppm. The D 2 O exchange experiments also verified the formation of carboxylic acid 9/unsubstituted acid amide 10. Additionally, the 13 C NMR showed a C 8 signal of C=O in 9 and 10, shifted upfield to 166.9 and 169.5 ppm, respectively, from the ketonic C 8 signal in 8 at 182.0 ppm. This was clear evidence for the substitution of the (COCCl 3 ) group, having a strong -I effect, by a group with a greater +R effect. A single (M) +• peak in the LR EIMS of 9 at 373 amu also assured the stability of the target molecule and the conversion of 8 to 9. The fragment 9a was produced after H 2 O loss and β-ketoacid-type rearrangement, which afforded a base peak at 355 amu (Scheme 3). The ultimate proof was provided by CHNS analysis, which showed comparable percentages for all elements. The trichlor-acetyl group, being a good leaving group, was conveniently substituted by OH or NH3 to produce indole acid 9 or amide 10, accordingly, by refluxing indole 8 with basified EtOH or excess aqueous NH3 in THF. The amide formation was authenticated by the appearance of two amide protons in the 1 H NMR. One proton emerged as a doublet (J = 3.0 Hz) at 7.81 ppm, and the other proton appeared as a singlet at 7.54 ppm. The D2O exchange experiments also verified the formation of carboxylic acid 9/unsubstituted acid amide 10. Additionally, the 13 C NMR showed a C 8 signal of C=O in 9 and 10, shifted upfield to 166.9 and 169.5 ppm, respectively, from the ketonic C 8 signal in 8 at 182.0 ppm. This was clear evidence for the substitution of the (COCCl3) group, having a strong -I effect, by a group with a greater +R effect. A single (M) +• peak in the LR EIMS of 9 at 373 amu also assured the stability of the target molecule and the conversion of 8 to 9. The fragment 9a was produced after H2O loss and β-ketoacid-type rearrangement, which afforded a base peak at 355 amu (Scheme 3). The ultimate proof was provided by CHNS analysis, which showed comparable percentages for all elements.   The trichlor-acetyl group, being a good leaving group, was conv by OH or NH3 to produce indole acid 9 or amide 10, accordingly, b with basified EtOH or excess aqueous NH3 in THF. The am authenticated by the appearance of two amide protons in the 1 H emerged as a doublet (J = 3.0 Hz) at 7.81 ppm, and the other proton a at 7.54 ppm. The D2O exchange experiments also verified the formatio 9/unsubstituted acid amide 10. Additionally, the 13 C NMR showed a C and 10, shifted upfield to 166.9 and 169.5 ppm, respectively, from the 8 at 182.0 ppm. This was clear evidence for the substitution of the (CO a strong -I effect, by a group with a greater +R effect. A single (M) +• p of 9 at 373 amu also assured the stability of the target molecule and th 9. The fragment 9a was produced after H2O loss and β-ketoacid-t which afforded a base peak at 355 amu (Scheme 3  The indole amide 10 was coupled with ester, methyl 2-bromopropanoate by deprotonating amidic and indolic NH of 10 with the subsequent addition of a coupling reagent to yield 11a. A strong base NaH in anhydrous DMSO was used for deprotonation, followed by refluxing of the reaction mixture at 170 • C for 2 h. The reaction was cooled before the addition of propanoate, followed by reflux. A mixture of products was obtained, rendered to column chromatography, and a white powder 12 was separated. The 1 H NMR of 12 ( Figure S7) showed all the expected aliphatic peaks and indole ring peaks, indicating the attachment of 10 with ester. It appeared that we had successfully synthesized 11a, but the 13 C NMR of 12 ( Figure S8) showed a few unjustified signals. The ESI MS of the product showed (M + H) +• at 473.1697 amu, which did not agree 11a with the expected (M) +• (426.46 amu for 11a) (Figure 2). before the addition of propanoate, followed by reflux. A mixture of products was obtained, rendered to column chromatography, and a white powder 12 was separated The 1 H NMR of 12 ( Figure S7) showed all the expected aliphatic peaks and indole ring peaks, indicating the attachment of 10 with ester. It appeared that we had successfully synthesized 11a, but the 13 C NMR of 12 ( Figure S8) showed a few unjustified signals. The ESI MS of the product showed (M + H) +• at 473.1697 amu, which did not agree 11a with the expected (M) +• (426.46 amu for 11a) ( Figure 2).  Figure 4 shows the optimized geometries of (10) and (12) generated through GaussView. These structures are true minima, as shown by the absence of any imaginary frequency. Comparing the calculated structure with the crystallographic information  Figure 4 shows the optimized geometries of (10) and (12) generated through GaussView. These structures are true minima, as shown by the absence of any imaginary frequency. Comparing the calculated structure with the crystallographic information shows that both results correlate well. For instance, in (12), C25-N3 is 1.365 (3) Å while C34-N6 is 1.310 (4) Å in the crystal structure. The computational bond lengths of these two bonds are 1.358 Å and 1.307 Å, respectively. Therefore, negligible deviations in computational results from the crystal structure can be seen. Such a comparison is not possible for compound (10) since we do not have its crystal structure.  Figure 4 shows the optimized geometries of (10) and (12) generated through GaussView. These structures are true minima, as shown by the absence of any imaginary frequency. Comparing the calculated structure with the crystallographic information shows that both results correlate well. For instance, in (12), C25-N3 is 1.365 (3) Å while C34-N6 is 1.310 (4) Å in the crystal structure. The computational bond lengths of these two bonds are 1.358 Å and 1.307 Å, respectively. Therefore, negligible deviations in computational results from the crystal structure can be seen. Such a comparison is not possible for compound (10) since we do not have its crystal structure.   (10) and (12) at PBE0-D3BJ/def2-TZVP level of theory. In 3D models, red represents oxygen, grey carbon, and blue nitrogen atoms. Hydrogen atoms are omitted for clarity. (Grey balls shows carbon atoms, red balls show oxygen, while show nitrogen).

Frontier Molecular Orbital (FMO) Analysis and Hyperpolarizability
Reactivity and other physical parameters, such as the energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbitals (LUMO), can be analyzed through frontier molecular orbital analysis [31]. In our case, since compound (10) exhibited an unexpected reactivity, its FMO analysis will be helpful in obtaining a better insight. Its calculated HOMO-LUMO energy difference is 4.51 eV. Figure 5 shows the iso-surface plots of HOMO and LUMO of compound (10). We can see that HOMO is present on the main indole framework, LUMO is on the right-side aromatic moiety, and the amido group is on the indole. Both compounds (10) and (12)  Reactivity and other physical parameters, such as the energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbitals (LUMO), can be analyzed through frontier molecular orbital analysis [31]. In our case, since compound (10) exhibited an unexpected reactivity, its FMO analysis will be helpful in obtaining a better insight. Its calculated HOMO-LUMO energy difference is 4.51 eV. Figure 5 shows the iso-surface plots of HOMO and LUMO of compound (10). We can see that HOMO is present on the main indole framework, LUMO is on the right-side aromatic moiety, and the amido group is on the indole. Both compounds (10) and (12) are expected to show excellent non-linear optical properties, as indicated by their hyperpolarizability (β) of 4643.02 a.u. and 1009.56 a.u., respectively. In the crystal structure of ( Figure 1 and Table 1), the 4,6-dimethoxy-1H-indole-7carbaldehyde group A (C7/C8/C15-C24/N1/O1-O3) is planar with root mean square

Crystal Data 2.3.1. Single Crystal XRD Depiction of 8 and 12
In the crystal structure of ( Figure 1 and Table 1), the 4,6-dimethoxy-1H-indole-7carbaldehyde group A (C7/C8/C15-C24/N1/O1-O3) is planar with root mean square deviation of 0.0426 Å. The phenyl rings B (C1-C6) and C (C9-C14) attached to group A are inclined at the dihedral angles of 7.39 (5) • and 59.16 (8) • . The bond lengths and angles of significant importance are consistent with the corresponding ones in the literature compounds [32,33]. The configuration of the molecule is stabilized by intra-molecular N-H· · · O bonding. Hydrogen bonding is not present in the supramolecular assembly. The molecules are connected through π· · · π stacking interaction along b-axis. The phenyl ring (C1-C6) of a molecule interacts with the 2,3-dihydro-1H-pyrrole and 1H-indole rings of the neighboring symmetry-related molecule with separation between the centers of interacting ring range from 4.13 to 4.18 Å ( Figure 6). The dihedral angles between the interacting rings range from 29.6 (4) • to 31.4 (3) • . The supramolecular assembly is further stabilized by weak C-H· · · π and C-Cl· · · π interactions with an H· · · π distance of 2.68 Å, whereas Cl· · · π has a distance of 5.718 (8) Å. In the crystal structure of ( Figure 3 and Table 1), the quinazoline ring A (C8-C13/C16/ C17/N1/N2) is planar with a root mean square deviation of 0.0207 Å. The methoxy group B (C14/O2) has a larger deviation from the plane of group A compared to the other methoxy group C (C15/O3). The benzaldehyde group D (C1-C7/O1) and phenyl ring E (C18-C23) are inclined at the dihedral angles of 9.96 (4) • and 10.59 (1) • with respect to group A. The methoxy group of methyl 2-hydroxypropanoate moiety F (C24-C26/C27A/O4/O5/O6A) is disordered over two sites and is stabilized by using various suitable restraints. In the crystal structure of ( Figure 3 and Table 1), the quinazoline ring A (C8-C13/C16/C17/N1/N2) is planar with a root mean square deviation of 0.0207 Å. The methoxy group B (C14/O2) has a larger deviation from the plane of group A compared to the other methoxy group C (C15/O3). The benzaldehyde group D (C1-C7/O1) and phenyl ring E (C18-C23) are inclined at the dihedral angles of 9.96 (4)° and 10.59 (1)° with respect to group A. The methoxy group of methyl 2-hydroxypropanoate moiety F (C24-C26/C27A/O4/O5/O6A) is disordered over two sites and is stabilized by using various suitable restraints.
The bond lengths and angles of significant importance are consistent with the corresponding ones in the literature compounds [34][35][36]. The molecules are connected in the form of a C(11) chain by C-H⋯O bonding, where CH is from ring E, and carbonyl Oatom of moiety F acts as an H-bond acceptor (Figure 7). The chain runs propagate along the b-axis. The supramolecular assembly is further stabilized by offset π⋯ π stacking interactions among the pyrimidine rings of the symmetry-related molecules and C-H⋯π interaction with CH from the methoxy group C (Figure 8). The dihedral angle between the symmetry-related pyrimidine rings is 0.03 (13)° with an inter-centroid separation of 4.27 Å. The bond lengths and angles of significant importance are consistent with the corresponding ones in the literature compounds [34][35][36]. The molecules are connected in the form of a C(11) chain by C-H· · · O bonding, where CH is from ring E, and carbonyl O-atom of moiety F acts as an H-bond acceptor (Figure 7). The chain runs propagate along the b-axis. The supramolecular assembly is further stabilized by offset π· · · π stacking interactions among the pyrimidine rings of the symmetry-related molecules and C-H· · · π interaction with CH from the methoxy group C (Figure 8). The dihedral angle between the symmetry-related pyrimidine rings is 0.03 (13) • with an inter-centroid separation of 4.27 Å.

Hirshfeld Surface Analysis
Supramolecular chemistry is deeply focused on understanding the interm interactions in the crystals, because these interactions are the decider of the prop this scenario, Hirshfeld surface analysis is performed using Crystal Explorer ver [37] to inspect the intermolecular interactions. The short contacts and comp longer contacts present in the crystal can be recognized by plotting Hirshfeld sur . The surface consists of red, blue and white colors. Red and blue regions sh Figure 8. Graphical illustration of π· · · π stacking and C-H· · · π interaction in the supramolecular assembly of 12. For clarity, H-atoms are not displayed.

Hirshfeld Surface Analysis
Supramolecular chemistry is deeply focused on understanding the intermolecular interactions in the crystals, because these interactions are the decider of the properties. In this scenario, Hirshfeld surface analysis is performed using Crystal Explorer version 21.5 [37] to inspect the intermolecular interactions. The short contacts and comparatively longer contacts present in the crystal can be recognized by plotting Hirshfeld surface over d norm . The surface consists of red, blue and white colors. Red and blue regions show short and long contacts, respectively. The contacts with a distance equal to the sum of the van der Waal radii are shown by white regions [38]. Figure 9a,b shows the Hirshfeld surface over d norm for 8 and 12, respectively. For 8, the red spot on the surface around carbonyl O-atom showed short O· · · Cl contact and red spots around CH of the methoxy group showed short H· · · C contacts ( Figure 9a). For 12, the red spot on the surface around carbonyl O-atom showed short O· · · H contact and red spots around CH of the phenyl ring showed short H· · · O contact ( Figure 9b). and long contacts, respectively. The contacts with a distance equal to the sum of the van der Waal radii are shown by white regions [38]. Figure 9a,b shows the Hirshfeld surface over for 8 and 12, respectively. For 8, the red spot on the surface around carbonyl O-atom showed short O⋯ Cl contact and red spots around CH of the methoxy group showed short H⋯ C contacts (Figure 9a). For 12, the red spot on the surface around carbonyl O-atom showed short O⋯H contact and red spots around CH of the phenyl ring showed short H⋯O contact (Figure 9b). The contribution of the contact to the stabilization of the supramolecular assembly of the crystal can be determined and displayed by 2D fingerprint plots [39]. Figure 10a-h show the 2D plots of significant importance for 8 and 12, respectively. For both compounds, H ⋯ H contacts make the largest contribution to the supramolecular The contribution of the contact to the stabilization of the supramolecular assembly of the crystal can be determined and displayed by 2D fingerprint plots [39]. Figure 10a-h show the 2D plots of significant importance for 8 and 12, respectively. For both compounds, H· · · H contacts make the largest contribution to the supramolecular assembly, with a percentage contribution of 32.4% in 8 (Figure 10a) and 49.2% in 12 (Figure 10e). The next important contact for 8 is H· · · Cl, with a contribution of 26.4% (Figure 11b), whereas the crystal structure of 12 contained no chlorine atoms, so there are no contacts involving the chlorine atom. O· · · H contact makes a larger contribution to 12 as compared to 8, as in the absence of chlorine atoms, oxygen atoms contribute more to the stabilization of supramolecular assembly. H· · · C contact makes a significant contribution to both compounds, as both have a number of short H· · · C contacts.  The crystal's response to applied stress depends on the voids present in it. A crystal with large cavities cannot bear a significant amount of stress. In order to check the mechanical strength of 8 and 12, we calculate voids using an approach reported in the literature [40]. The pro-crystal electron density idea is used to calculate voids with an isosurface of 0.0002 a.u (Figure 11). The volume of voids is 334.69 and 399.09 Å 3 in 8 and 12, respectively. The voids consume 15.1% of the space in 8 and 16.4% in 12. The voids occupied a small space in both compounds, so there is no large cavity, and compounds are expected to have a good mechanical response.

In Silico Anticholinesterase Activity
Virtual screening of the proposed compounds (5-10 and 12) was performed against  The crystal's response to applied stress depends on the voids present in it. A crystal with large cavities cannot bear a significant amount of stress. In order to check the mechanical strength of 8 and 12, we calculate voids using an approach reported in the literature [40]. The pro-crystal electron density idea is used to calculate voids with an isosurface of 0.0002 a.u (Figure 11). The volume of voids is 334.69 and 399.09 Å 3 in 8 and 12, respectively. The voids consume 15.1% of the space in 8 and 16.4% in 12. The voids occupied a small space in both compounds, so there is no large cavity, and compounds are expected to have a good mechanical response.

In Silico Anticholinesterase Activity
Virtual screening of the proposed compounds (5-10 and 12) was performed against The crystal's response to applied stress depends on the voids present in it. A crystal with large cavities cannot bear a significant amount of stress. In order to check the mechanical strength of 8 and 12, we calculate voids using an approach reported in the literature [40]. The pro-crystal electron density idea is used to calculate voids with an iso-surface of 0.0002 a.u (Figure 11). The volume of voids is 334.69 and 399.09 Å 3 in 8 and 12, respectively. The voids consume 15.1% of the space in 8 and 16.4% in 12. The voids occupied a small space in both compounds, so there is no large cavity, and compounds are expected to have a good mechanical response.

In Silico Anticholinesterase Activity
Virtual screening of the proposed compounds (5-10 and 12) was performed against Human Acetylcholinesterase (hAChE). All compounds show very good binding to hAChE. A dimeric form of the enzyme was used for the docking study, and it was very interesting to note that 6 and 10 showed binding to the dimeric interface of the enzyme (ALA377  Table 2).   All other ligands preferably bind to the peripheral anionic site (PAS) of acetylcholinesterase, consisting of TYR72, ASP74, TYR124, TRP286, and TYR341 amino acids. The PAS site is connected to the active site residue of the enzyme SER203, GLU334, and HIS447 by a narrow path. Ligand 7 shows the best binding to PAS residue, including TYR72, ASP74, THR75, LE 76, THR83, TRP286, HIS287, LEU289, GLN291, GLU292, SER293, VAL294, PHE295, ARG296, PHE297, TYR337, PHE338, TYR341, GLY342 ( Figure  12) with binding energy and dissociation constant of −11.13 kcal/mol and 6.97 nM, respectively (Table 2). Four H-bonds were observed, i.e., two with TYR72 (3.3 and 3.45 Å), one with each THR75 (3.08 Å) and ARG296 (3.48 Å) (Figure 12). A π-π stacking was also observed with TRP286, which has been reported to impair enzyme activity. The PHE297 from the acyl pocket of the enzyme is also surrounded by the ligand and may involve in All other ligands preferably bind to the peripheral anionic site (PAS) of acetylcholinesterase, consisting of TYR72, ASP74, TYR124, TRP286, and TYR341 amino acids. The PAS site is connected to the active site residue of the enzyme SER203, GLU334, and HIS447 by a narrow path. Ligand 7 shows the best binding to PAS residue, including TYR72, ASP74, THR75, LE 76, THR83, TRP286, HIS287, LEU289, GLN291, GLU292, SER293, VAL294, PHE295, ARG296, PHE297, TYR337, PHE338, TYR341, GLY342 ( Figure 12) with binding energy and dissociation constant of −11.13 kcal/mol and 6.97 nM, respectively (Table 2). Four H-bonds were observed, i.e., two with TYR72 (3.3 and 3.45 Å), one with each THR75 (3.08 Å) and ARG296 (3.48 Å) ( Figure 12). A π-π stacking was also observed with TRP286, which has been reported to impair enzyme activity. The PHE297 from the acyl pocket of the enzyme is also surrounded by the ligand and may involve in impairing its interactions with its substrate acetyl group. Previously, TRP286 and VAL294 were found to interact with substituted benzene rings in azine derivatives by π-πand π-alkyl hydrophobic interactions [41].

Anticholinesterase and Antibacterial Activities
Azine derivatives, benzoxazepines and benzodiazepines show various biological activities, including antibacterial, antifungal, and anticholinesterase activities [42][43][44]. This makes benzo-diazine an excellent candidate for exploration of its potential for cholinesterase inhibition and antimicrobial activities. The synthesized compounds (8, 9, 10 and 12) were subjected to anti-acetylcholinesterase activity, and 8 and 12 showed better acetylcholinesterase inhibition in comparison to other compounds, showing IC 50 values of 7.31 and 6.11 µM, respectively, but due to solubility issues the IC 50 values can be far less than the apparent value. The same can be true for 9 and 12, precipitated even at lower concentrations in the reaction mixture. The standard drug galantamine IC 50 value was 5.32 µM. The in silico results also agree with these findings and show very low dissociation constants and strong binding energies. In addition, 4,6-Dimethoxyindole-based Azines derivative showed a maximum of 30-64% inhibition of acetylcholinesterase while using 200 µM compounds, where dimethoxy substituted compounds were shown to be the best inhibitor [41]. Some azine Schiff bases showed an IC 50 of 23.60 ± 0.63 µg/mL and 28.59 ± 0.07 µg/mL [45].

Antibacterial and Anticholinesterase Activities
Antibacterial activity was assessed against clinically isolated, antibiotic-resistant strains of Salmonella typhi and Staphylococcus aureus using Disk diffusion assay, as previously described [62]. Anticholinesterase activity was also performed, as described earlier for indoles.

Single Crystal XRD Details of 10 and 12
The crystals of suitable size are selected and mounted on Bruker Kappa Apex-II for the sake of data collection, as molybdenum X-ray source. For the structure solution and refinement, SHELXT-2014 [61] and SHELXL-2019/2 [63] software were used. Nonhydrogen atoms and H-atoms were assigned anisotropic displacement (parameters and isotropic displacement parameters, respectively). For eye-catching graphics, PLATON [64] and Mercury [65,66] software were employed.

Conclusions
During trials to synthesize pharmacologically potent indole fused benoxazepine/ benzodiazepine heterocycles, an unexpectedly rearranged novel product, 1,3-bezodiazine, was obtained. Compounds (10) and (12) were subject to computational insight (DFT studies and Hirshfeld surface charge analysis) for a better understanding of geometric features and reactivity of the products, to create an idea for the design of derivative drugs from the novel compound. FMO analysis revealed that LUMO is present over amido function, which is expected to undergo internal rearrangement. An excellent nonlinear optical response is expected as a result of significant hyperpolarizability. The synthesized compounds, particularly 3-bezodiazine, have shown considerable anticholinesterase and antibacterial activities, and these can be explored further for in vitro and in vivo activities and toxicity at the cellular level.