Synthesis of Novel Nitro-Halogenated Aryl-Himachalene Sesquiterpenes from Atlas Cedar Oil Components: Characterization, DFT Studies, and Molecular Docking Analysis against Various Isolated Smooth Muscles

We report the synthesis of two novel halogenated nitro-arylhimachalene derivatives: 2-bromo-3,5,5,9-tetramethyl-1-nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulene (bromo-nitro-arylhimachalene) and 2-chloro-3,5,5,9-tetramethyl-1,4-dinitro-6,7,8,9-tetrahydro-5H-benzo[7]annulene (chloro-dinitro-arylhimachalene). These compounds were derived from arylhimachalene, an important sesquiterpene component of Atlas cedar essential oil, via a two-step halogenation and nitration process. Characterization was performed using 1H and 13C NMR spectrometry, complemented by X-ray structural analysis. Quantum chemical calculations employing density functional theory (DFT) with the Becke3-Lee-Yang-parr (B3LYP) functional and a 6-31++G(d,p) basis set were conducted. The optimized geometries of the synthesized compounds were consistent with X-ray structure data. Frontier molecular orbitals and molecular electrostatic potential (MEP) profiles were identified and discussed. DFT reactivity indices provided insights into the compounds’ behaviors. Moreover, Hirshfeld surface and 2D fingerprint analyses revealed significant intermolecular interactions within the crystal structures, predominantly H–H and H–O contacts. Molecular docking studies demonstrate strong binding affinities of the synthesized compounds to the active site of protein 7B2W, suggesting potential therapeutic applications against various isolated smooth muscles and neurotransmitters.


Introduction
The essential oil of Atlas cedar, Cedrus atlantica, is essentially composed of sesquiterpenic hydrocarbons α-, β-, and γ-himachalene (Figure 1) and together they can make up to 70% of its contents [1].This oil can be explored as a renewable feedstock for several industrial processes and pharmaceutical compounds.Arylhimachalene is another minor constituent of this oil that can easily be obtained by aromatizing the α-, β-, and γ-himachalene mixture [2].Structurally, arylhimachalene is a natural benzocylcohetane; this structure motif is present in numerous important medicines (desloratadine [3] and amitriptyline [4]) (Figure 2) and natural products (colchicine and phomarol) (Figure 2).It is an important building block for the synthesis of biologically active compounds.Thus, we are interested in himachalenes, and particularly in arylhimachalene, as a readily available starting material for the synthesis of novel biologically active benzocycloheptanic deriva-

Introduction
The essential oil of Atlas cedar, Cedrus atlantica, is essentially composed of ses erpenic hydrocarbons α-, β-, and γ-himachalene (Figure 1) and together they can ma to 70% of its contents [1].This oil can be explored as a renewable feedstock for se industrial processes and pharmaceutical compounds.Arylhimachalene is another m constituent of this oil that can easily be obtained by aromatizing the α-, β-, and γ-h chalene mixture [2].Structurally, arylhimachalene is a natural benzocylcohetane structure motif is present in numerous important medicines (desloratadine [3] and triptyline [4]) (Figure 2) and natural products (colchicine and phomarol) (Figure 2) an important building block for the synthesis of biologically active compounds.Thu are interested in himachalenes, and particularly in arylhimachalene, as a readily ava starting material for the synthesis of novel biologically active benzocycloheptanic de tives of himachalenes.In fact, we have previously reported the synthesis and mole docking analysis of trans-himachalol, a synthetic isomer of the naturally occurring quiterpene cis-himachalol; the synthesized compound has shown promising acti against various isolated smooth muscles and against different neurotransmitter Moreover, arylhimachalene derivatives have been previously reported to exhibit diff biological activities [6][7][8].Nitroarenes are a well-known compound with versatile applications from perf and pharmaceuticals to dyes and explosives [9][10][11].They can be obtained by an ele philic hydrogen substitution [12][13][14].The reactivity of the aromatic compounds dep on the substituted groups.They can either activate or deactivate the arenes.Substi groups can also act as directing agents, favoring one regioisomer over another [15].
In order to enhance the previously reported promising biological activities of h chalenes derivatives [6][7][8], herein, we report the syntheses and the struc 7B2W, suggesting potential therapeutic applications against various isolated smooth muscles and neurotransmitters.

Introduction
The essential oil of Atlas cedar, Cedrus atlantica, is essentially composed of sesquiterpenic hydrocarbons α-, β-, and γ-himachalene (Figure 1) and together they can make up to 70% of its contents [1].This oil can be explored as a renewable feedstock for several industrial processes and pharmaceutical compounds.Arylhimachalene is another minor constituent of this oil that can easily be obtained by aromatizing the α-, β-, and γ-himachalene mixture [2].Structurally, arylhimachalene is a natural benzocylcohetane; this structure motif is present in numerous important medicines (desloratadine [3] and amitriptyline [4]) (Figure 2) and natural products (colchicine and phomarol) (Figure 2).It is an important building block for the synthesis of biologically active compounds.Thus, we are interested in himachalenes, and particularly in arylhimachalene, as a readily available starting material for the synthesis of novel biologically active benzocycloheptanic derivatives of himachalenes.In fact, we have previously reported the synthesis and molecular docking analysis of trans-himachalol, a synthetic isomer of the naturally occurring sesquiterpene cis-himachalol; the synthesized compound has shown promising activities against various isolated smooth muscles and against different neurotransmitters [5].Moreover, arylhimachalene derivatives have been previously reported to exhibit different biological activities [6][7][8].Nitroarenes are a well-known compound with versatile applications from perfumes and pharmaceuticals to dyes and explosives [9][10][11].They can be obtained by an electrophilic hydrogen substitution [12][13][14].The reactivity of the aromatic compounds depends on the substituted groups.They can either activate or deactivate the arenes.Substituted groups can also act as directing agents, favoring one regioisomer over another [15].
In order to enhance the previously reported promising biological activities of himachalenes derivatives [6][7][8], herein, we report the syntheses and the structural Nitroarenes are a well-known compound with versatile applications from perfumes and pharmaceuticals to dyes and explosives [9][10][11].They can be obtained by an electrophilic hydrogen substitution [12][13][14].The reactivity of the aromatic compounds depends on the substituted groups.They can either activate or deactivate the arenes.Substituted groups can also act as directing agents, favoring one regioisomer over another [15].

Results and Discussion
2-bromo-1-ntitro-arylhimachalene (4) and 2-chloro-1,4-dintitro-arylhimachalene (5) were prepared from arylhimachalene in a two-step process.The first step is the halogenation of arylhimachalene, and the second step is the nitration of the halogenated products by a mixture of nitric acid and sulfuric acid.
The treatment of arylhimachalene with N-chlorosuccinimide (NCS) took place in acetonitrile at 80 • C [16] and led to the formation of chloro-arylhimachalene (3) with an 85% yield (Scheme 1).The absence of any benzylic chlorination products even with the use of two equivalents of NCS suggests that the reaction likely proceeds via an electrophilic substitution mechanism rather than a radical one [23].

Results and Discussion
2-bromo-1-ntitro-arylhimachalene (4) and 2-chloro-1,4-dintitro-arylhimachalene were prepared from arylhimachalene in a two-step process.The first step is the halog ation of arylhimachalene, and the second step is the nitration of the halogenated prod by a mixture of nitric acid and sulfuric acid.
The treatment of arylhimachalene with N-chlorosuccinimide (NCS) took place in etonitrile at 80 °C [16] and led to the formation of chloro-arylhimachalene (3) with an yield (Scheme 1).The absence of any benzylic chlorination products even with the us two equivalents of NCS suggests that the reaction likely proceeds via an electrophilic s stitution mechanism rather than a radical one [23].The good results obtained for the chloration of arylhimachalene with NCS enco aged us to use N-bromosuccinimide (NBS) for the bromination.The use of NBS led total conversion but an unsatisfying selectivity due to the formation of other multi mated products like the (Z)-2,8-dibromo-9-(bromomethylene)-3,5,5-trimethyl-6,7,8,9 rahydro-5H-benzo [7]annulene that can be obtained using the same protocol [24].To hance selectivity, arylhimachalene was subjected to a HBr/H2O2 treatment.This appro was highly effective, achieving complete conversion of arylhimachalene with 100% se tivity for the target product 2 (Scheme 1).

Nitration of Arylhimachalane-Halogenated Derivatives
While treating the bromo-arylhimachalene with a mixture of nitric acid and sulf acid led to a total conversion, giving the nitro derivative (4) an 83% yield, treating chl arylhimachalene with the same acid mixture gave chloro-dinitro-arylhimachalene (5 stead of the mono-nitrated derivative with a 79% yield (Scheme 2).The good results obtained for the chloration of arylhimachalene with NCS encouraged us to use N-bromosuccinimide (NBS) for the bromination.The use of NBS led to a total conversion but an unsatisfying selectivity due to the formation of other multibromated products like the (Z)-2,8-dibromo-9-(bromomethylene)-3,5,5-trimethyl-6,7,8,9-tetrahydro-5H-benzo [7]annulene that can be obtained using the same protocol [24].To enhance selectivity, arylhimachalene was subjected to a HBr/H 2 O 2 treatment.This approach was highly effective, achieving complete conversion of arylhimachalene with 100% selectivity for the target product 2 (Scheme 1).

Nitration of Arylhimachalane-Halogenated Derivatives
While treating the bromo-arylhimachalene with a mixture of nitric acid and sulfuric acid led to a total conversion, giving the nitro derivative (4) an 83% yield, treating chloro-arylhimachalene with the same acid mixture gave chloro-dinitro-arylhimachalene (5) instead of the mono-nitrated derivative with a 79% yield (Scheme 2).Scheme 2. Nitration of bromo-arylhimachalene and chloro-arylhimachalene.

Structural Description of the Compounds
The compound crystallizes in the orthorhombic system with the P212121 space group.The molecular structure of this compound is illustrated in Figure 3.The asymmetric unit comprises two distinct molecules exhibiting different conformations, which represents the automatic fit of the two crystallographically independent molecules, as illustrated in Figure 4 [25].Each molecule is built up from fused six-and seven-membered rings.In the first molecule (C1-C15), the seven-membered ring shows a chair conformation, whereas in the second molecule (C16-C30), the seven-membered ring displays a screw-boat conformation.The plane passing through the nitro group (O1O2N1C11) is almost perpendicular to the benzene ring (C1 to C11), as indicated by the value of the dihedral angle of (O1O2N1C11)/(C1 to C11), which is equal to 93.12°, while the plane passing through the nitro group (O3O4N2C26) is slightly offset from the perpendicular to the benzene ring, as shown by the value of the dihedral angle (O3O4N2C26)/(C16 to C26), which is equal to 95.85°; Figure 5 shows the two dihedral angles.Table 1 summarizes the crystallographic data, experimental details of data collection, and structure refinements.The crystal structure's interlayer connectivity is maintained through hydrogen bonds (C30-H30A… O2 i ), involving H atoms from methyl groups on benzene rings and offset π…π interactions between aromatic rings.These interactions are illustrated in Figures 6 and 7, with a centroid-to-centroid distance of 4.818 (4) Å.Additionally, the crystal structure exhibits C4-H4B… Cg1 ii interactions, where Cg represents the centroid of the benzene rings.Collectively, these interactions create a three-dimensional network, depicted in Figure 8.The compound crystallizes in the orthorhombic system with the P2 1 2 1 2 1 space group.The molecular structure of this compound is illustrated in Figure 3.The asymmetric unit comprises two distinct molecules exhibiting different conformations, which represents the automatic fit of the two crystallographically independent molecules, as illustrated in Figure 4 [25].Each molecule is built up from fused six-and seven-membered rings.

D-H•••
In the first molecule (C1-C15), the seven-membered ring shows a chair conformation, whereas in the second molecule (C16-C30), the seven-membered ring displays a screwboat conformation.The plane passing through the nitro group (O1O2N1C11) is almost perpendicular to the benzene ring (C1 to C11), as indicated by the value of the dihedral angle of (O1O2N1C11)/(C1 to C11), which is equal to 93.12 • , while the plane passing through the nitro group (O3O4N2C26) is slightly offset from the perpendicular to the benzene ring, as shown by the value of the dihedral angle (O3O4N2C26)/(C16 to C26), which is equal to 95.85 • ; Figure 5 shows the two dihedral angles.Table 1 summarizes the crystallographic data, experimental details of data collection, and structure refinements.The crystal structure's interlayer connectivity is maintained through hydrogen bonds (C30-H30A. . .O2 i ), involving H atoms from methyl groups on benzene rings and offset π. ..π interactions between aromatic rings.These interactions are illustrated in Figures 6 and 7, with a centroid-to-centroid distance of 4.818 (4) Å.Additionally, the crystal structure exhibits C4-H4B. . .Cg1 ii interactions, where Cg represents the centroid of the benzene rings.Collectively, these interactions create a three-dimensional network, depicted in Figure 8.

2-Chloro-1:3-Dintitro-Arylhimachalene
The synthesized compound C 15 H 19 ClN 2 O 4 consists of two fused rings: a six-membered and seven-membered ring.The seven-membered ring adopts a screw-boat conformation characterized by the following parameters: QT = 0.9266 (2) Å, θ = 68.85(2) • , φ2 = −40.54(1) • , and φ3 = 166.87(3) • [26].The presence of the Cl atom allows for the absolute configuration to be definitively determined as C2(S) [27].The bond length of C10-Cl1 is 1.73 Å, which aligns well with the reported value of 1.738 (2) for a similar bond between a benzene ring and a chlorine atom [28].The molecular structure is illustrated in Figure 9.The plane formed by the nitro group (O3O4N2C11) is nearly perpendicular to the benzene ring, with a dihedral angle of 92.06 • , while the dihedral angle between the nitro group (O1O2N1C8) and the benzene ring is 105.45 • .Figure 10 illustrates these two dihedral angles.The crystal packing does not exhibit any specific intermolecular interactions.

DFT Computation and Electronic Structure
To gain a deeper understanding of the electronic structure, the optimization of compounds 4 and 5 was performed using the DFT/B3LYP method, focusing on the singlet ground state.Figure 11 shows the optimized geometry of the two molecules, while Table

DFT Computation and Electronic Structure
To gain a deeper understanding of the electronic structure, the optimization of compounds 4 and 5 was performed using the DFT/B3LYP method, focusing on the singlet ground state.Figure 11 shows the optimized geometry of the two molecules, while Table

DFT Computation and Electronic Structure
To gain a deeper understanding of the electronic structure, the optimization of compounds 4 and 5 was performed using the DFT/B3LYP method, focusing on the singlet ground state.Figure 11 shows the optimized geometry of the two molecules, while Table 2 lists the selected structural parameters, bond lengths, bond angles, and torsion angles, with respect to the most stable conformer in the optimized structure and the single crystal.The data presented in Table 2 show that the geometrical parameters for the synthesized compounds 4 and 5 exhibit a high degree of concordance between the experimental and theoretical results.The majority of the calculated values are in close alignment with the experimental data, demonstrating no notable discrepancies between the single crystal and optimized structures of these stable compounds (4 and 5).The computed geometrical parameters excellently coincide with the experimental data.However, in a few cases, slight mismatches can be attributed to phase differences between the experiment and computation, as well as to the absence of packing effects in the gas-optimized structure.Over-  The data presented in Table 2 show that the geometrical parameters for the synthesized compounds 4 and 5 exhibit a high degree of concordance between the experimental and theoretical results.The majority of the calculated values are in close alignment with the experimental data, demonstrating no notable discrepancies between the single crystal and optimized structures of these stable compounds (4 and 5).The computed geometrical parameters excellently coincide with the experimental data.However, in a few cases, slight mismatches can be attributed to phase differences between the experiment and computation, as well as to the absence of packing effects in the gas-optimized structure.Overall, these results indicate a strong concurrence between the experimental findings and those obtained through the DFT calculations.

Frontier Molecular Orbital (FMO) and Reactivity Parameter Analysis
The theoretical calculations yielded valuable insights into the energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).These frontier orbitals are crucial for demonstrating a molecule's electron-donating or electron-accepting properties.Furthermore, they serve as a foundation for calculating various reactivity parameters according to Koopman's theorem [29].Global reactivity parameters are pivotal for determining the reactivity profiles of organic compounds, offering a deeper understanding of their chemical reactivity and kinetic stability, as well as their electronic behavior during chemical processes.Key parameters include the chemical potential (µ), chemical hardness (η), electronegativity (χ), and electrophilicity index (ω).The calculated values of these electronic quantities characterizing the molecular structures are listed in Table 3. Compounds exhibiting a small energy difference between the HOMO and LUMO (∆Egap) and high softness (σ) demonstrate heightened reactivity.This leads to stronger interactions when a target molecule with high HOMO energy acts as an electron donor, interacting with a molecule serving as an acceptor with lower LUMO energy.
The calculated energy gaps for the synthesized compounds 4 and 5 are 4.351 eV and 4.526 eV, respectively.These values suggest that the compounds exhibit low kinetic stability and high chemical reactivity, making them good precursors for further development and synthesis of more complex molecules.
Notably, compound 5 displays lower energy orbitals than compound 4, indicating enhanced reactivity in the latter.This behavior is further supported by the observed values of chemical potential and hardness.Compound 4, with a high electrophilicity index of 3.013 eV, is identified as a potent electrophile, thereby acting as a more effective electron acceptor than compound 5.
For compound 4, the HOMO is primarily localized on the two double bonds C1=C14 and C17=C18, as well as on the bromine atom.However, no electron density is observed on the C15 and C19 atoms.Conversely, the LUMO is predominantly located in the NO 2 group.In the HOMO-1 orbital, the electron density is mainly concentrated on the phenyl ring, particularly on the double bonds C15=C17, C1=C19, and C18=C19, and to a lesser extent on the bromine atom (Figure 12).The molecular orbitals of the C1=C14 and C17=C18 double bonds remain devoid of any electron density.An orbital contribution investigation indicates that the composition of HOMO comes mainly from the orbitals of C1, C14, C17, and C18 and Br39 atoms.In contrast, the components of LUMO come mainly from the nitro moiety (Table 4).
In compound 5, the HOMO orbitals are primarily situated on the benzene ring, specifically on the two double bonds C1=C14 and C16=C17, as well as on the chlorine atom (Figure 12).In contrast, the NO 2 groups exhibit a lack of electron density.The composition of HOMO comes mainly from the atomic orbitals of C1, C14, C16, C17, and Cl 35.As for the LUMO orbital, it is located on the C14=C15 and C17=C18 bonds and on one of the two NO 2 groups (Table 4).

Molecular Electrostatic Potential (MEP)
The molecular electrostatic potential (MEP) has proven to be a valuable tool for investigating the correlation between molecular structure and physicochemical properties [30,31].The MEP map is a valuable tool for evaluating the reactivity of both inorganic and organic molecules, as well as for identifying potential internal and external molecular interactions.It indicates that areas with the highest positive potential, typically represented in blue, are likely targets for nucleophilic attack.Conversely, areas with the highest negative potential, shown in red, are susceptible to electrophilic attack.When analyzing the MEP map of the two compounds, we observe that the negatively charged regions are largely located over the oxygen atoms of the nitro moiety (Figure 13).These are the sites of strong electrophilic reactivity for the compound.Bromine and chlorine atoms are located in regions characterized by medium electron density, which means that bromine and chlorine are less likely to react either as nucleophiles or electrophiles.On the other hand, the positive regions are mainly surrounding the molecule over the carbons (aromatic CH 3 for compound 5) and hydrogen atoms, showing electrophilic reactivity.
In compound 5, the HOMO orbitals are primarily situated on the benzene ring, specifically on the two double bonds C1=C14 and C16=C17, as well as on the chlorine atom (Figure 12).In contrast, the NO2 groups exhibit a lack of electron density.The composition of HOMO comes mainly from the atomic orbitals of C1, C14, C16, C17, and Cl 35.As for the LUMO orbital, it is located on the C14=C15 and C17=C18 bonds and on one of the two NO2 groups (Table 4).

Molecular Electrostatic Potential (MEP)
The molecular electrostatic potential (MEP) has proven to be a valuable tool for investigating the correlation between molecular structure and physicochemical properties [30,31].The MEP map is a valuable tool for evaluating the reactivity of both inorganic and organic molecules, as well as for identifying potential internal and external molecular interactions.It indicates that areas with the highest positive potential, typically represented in blue, are likely targets for nucleophilic attack.Conversely, areas with the highest negative potential, shown in red, are susceptible to electrophilic attack.When analyzing the MEP map of the two compounds, we observe that the negatively charged regions are largely located over the oxygen atoms of the nitro moiety (Figure 13).These are the sites of strong electrophilic reactivity for the compound.Bromine and chlorine atoms are located in regions characterized by medium electron density, which means that bromine and chlorine are less likely to react either as nucleophiles or electrophiles.On the other hand, the positive regions are mainly surrounding the molecule over the carbons (aromatic CH3 for compound 5) and hydrogen atoms, showing electrophilic reactivity.

Hirshfeld Surface Analyses
The packing of small molecule organic crystals is primarily governed by hydrogen bonding patterns.However, the crystalline structure is the result of multiple forces,

Hirshfeld Surface Analyses
The packing of small molecule organic crystals is primarily governed by hydrogen bonding patterns.However, the crystalline structure is the result of multiple forces, necessitating the consideration of all intermolecular interactions.The unique characteristics of crystal packing arise from the molecule's ability to engage in specific intermolecular interactions.Similarly, the reactivity of compounds is influenced by intermolecular interactions in a reaction medium.Hence, the analysis of intermolecular interactions is crucial.An interesting tool for studying these interactions in the crystalline phase is to investigate Hirshfeld surfaces and 2D fingerprints generated by the CrystalExplorer program [32].These surfaces were developed to define the molecule's spatial occupancy within the crystal and facilitate the partitioning of the crystal's electron density into molecular fragments.Short contacts are visually highlighted in red on Hirshfeld surfaces, whereas blue areas indicate longer distances.
Figure 14 shows the Hirshfeld surfaces plotted over d norm for compounds 4 and 5, along with the fingerprint plot of their intermolecular contacts shown in Figure 15.The analysis of these surfaces and fingerprint plots reveals significant intermolecular interactions in both crystal packings.In the compound 4 crystal packing, the most prevalent interactions are H. ..H (50.4%) and H. ..O (22.1%).Similarly, the compound 5 crystal packing is dominated by H. ..H interactions at 36.2%, followed by H. ..O at 35.3% (Figure 15).Additionally, H. ..Br and H. ..Cl interactions are present, accounting for 13.2% and 12% of the total Hirshfeld surface, respectively.Importantly, the absence of long sharp spikes indicates the lack of strong hydrogen bonds in the crystal structures of the title compounds (Figure 15).along with the fingerprint plot of their intermolecular contacts shown in Figure 15.The analysis of these surfaces and fingerprint plots reveals significant intermolecular interactions in both crystal packings.In the compound 4 crystal packing, the most prevalent interactions are H…H (50.4%) and H…O (22.1%).Similarly, the compound 5 crystal packing is dominated by H…H interactions at 36.2%, followed by H…O at 35.3% (Figure 15).Additionally, H…Br and H…Cl interactions are present, accounting for 13.2% and 12% of the total Hirshfeld surface, respectively.Importantly, the absence of long sharp spikes indicates the lack of strong hydrogen bonds in the crystal structures of the title compounds (Figure 15).analysis of these surfaces and fingerprint plots reveals significant intermolecular interactions in both crystal packings.In the compound 4 crystal packing, the most prevalent interactions are H…H (50.4%) and H…O (22.1%).Similarly, the compound 5 crystal packing is dominated by H…H interactions at 36.2%, followed by H…O at 35.3% (Figure 15).Additionally, H…Br and H…Cl interactions are present, accounting for 13.2% and 12% of the total Hirshfeld surface, respectively.Importantly, the absence of long sharp spikes indicates the lack of strong hydrogen bonds in the crystal structures of the title compounds (Figure 15).

Molecular Docking Analysis
Molecular docking is an essential method used in computational drug design and screening processes.It enables the evaluation of the interaction between a molecule and a receptor by determining the binding affinity score [33].In this research, we explored the potential of compounds synthesized from the synthesis of two halogenated nitro derivatives of arylhimachalene: bromo-nitro-arylhimachalene and chloro-dinitro-arylhimachalene, using molecular docking.These compounds exhibited notable efficacy in modulating the activity of assorted isolated smooth muscles and in interactions with diverse neurotransmitters.Docking scores were employed to evaluate the binding affinity strength in ligand-receptor interactions, where a lower (more negative) score signifies a more robust association between the target and ligand (Table 5).
The findings reveal that the bromo-nitro-arylhimachalene and chloro-dinitro-arylhimachalene compounds exhibited significant binding affinities to the active site of protein 7B2W.The estimated binding energies were −6.211 kcal/mol and −6.967 kcal/mol, respectively, which significantly exceeded the binding energy of the positive control, physostigmine, recorded at 3.950 kcal/mol in this research.The consistency between our results and those reported in the literature is notable.In previous studies, himachalone monohydrochloride demonstrated binding energies of −5.798 kcal/mol, trans-himachalol showed −7.137 kcal/mol, and himachalone −7.049 kcal/mol [5].These findings reinforce the validity of our research outcomes, suggesting that our compounds possess comparable or even superior inhibitory potential.Such consistency across different studies underscores the robustness of our experimental methodology and the reliability of our results.
Table 5.The docking scores (kcal/mol) and detailed interaction studies of the selected compounds against the protein 7B2W.

Compound Docking Score Interacting Amino Acid Interaction Type
Bromo-nitro-arylhimachalene −6.579While the molecular docking process commonly faces challenges related to efficient sampling of conformations and orientations, ensuring that ligand conformations maintain low energy levels is occasionally disregarded.However, this aspect is crucial, as highenergy conformations can serve as decoys, potentially masking the presence of more favorable poses [34].Neglecting electrostatic energies in ligand conformations is one factor that can contribute to such high-energy states.Often, these energies are overlooked due to concerns about their potential overemphasis in calculations employing effectively low dielectric environments [35] (Figure 16).low energy levels is occasionally disregarded.However, this aspect is crucial, as highenergy conformations can serve as decoys, potentially masking the presence of more favorable poses [34].Neglecting electrostatic energies in ligand conformations is one factor that can contribute to such high-energy states.Often, these energies are overlooked due to concerns about their potential overemphasis in calculations employing effectively low dielectric environments [35] (Figure 16).Further investigations were conducted to determine the specific interactions of the more stable compounds at the protein's active site and to clarify the mechanism of inhibition.As depicted in Figure 17, bromo-nitro-arylhimachalene and chloro-dinitroarylhimachalene, which displayed the lowest binding energy and are considered the most Further investigations were conducted to determine the specific interactions of the more stable compounds at the protein's active site and to clarify the mechanism of inhibition.As depicted in Figure 17, bromo-nitro-arylhimachalene and chloro-dinitro-arylhimachalene, which displayed the lowest binding energy and are considered the most probable active inhibitors compared to the control, interacted with the surrounding residues through various interactions.Experimental validation is essential to assess the therapeutic effects and practical applications of promising compounds.The molecular docking results provide insights, but in vitro and in vivo experiments are necessary for a comprehensive evaluation.These experiments will elucidate interactions with target receptors and enzymes, pharmacokinetics, safety, and efficacy profiles.Such an investigation is crucial for advancing the halogenated nitro derivatives of arylhimachalene as potential drugs for medical treatments [36].
therapeutic effects and practical applications of promising compounds.The molecular docking results provide insights, but in vitro and in vivo experiments are necessary for a comprehensive evaluation.These experiments will elucidate interactions with target receptors and enzymes, pharmacokinetics, safety, and efficacy profiles.Such an investigation is crucial for advancing the halogenated nitro derivatives of arylhimachalene as potential drugs for medical treatments [36].

Materials
All chemicals and solvents were purchased from Sigma-Aldrich and were utilized as received, without additional purification.The 1 H and 13 C NMR spectra were acquired using a Bruker Avance 300 spectrometer operating at 300 MHz for 1 H and 75 MHz for 13 C (University of Colorado Boulder, Boulder, CO, USA), with tetramethylsilane serving as the internal standard.Chemical shifts were reported in δ values (ppm).X-ray diffraction analyses were performed on a Bruker D8 VENTURE Super DUO diffractometer (Bruker, Billerica, MA, USA).

Procedure for the Synthesis of Compound 2
In a 100 mL round-bottom flask equipped with a magnetic stirrer, 1 g (4.901 mmol) of arylhimachalene and 1.42 g (17.5 mmol) of HBr were introduced.Then, 0.5 mL of H2O2 was added dropwise over 1 h.The reaction mixture was stirred for 24 h at 0 °C.At the end of the reaction, monitored by thin-layer and gas chromatography, the mixture was neutralized by a saturated aqueous solution of NaHCO3, extracted with ethyl acetate (3 × 20 mL).The organic layers were gathered and dried over anhydrous magnesium sulfate (MgSO4) and filtered.The solvent was removed in vacuo.The residue was purified by column chromatography using silica gel with hexane/AcOEt (98:2) as the eluent.

Materials
All chemicals and solvents were purchased from Sigma-Aldrich and were utilized as received, without additional purification.The 1 H and 13 C NMR spectra were acquired using a Bruker Avance 300 spectrometer operating at 300 MHz for 1 H and 75 MHz for 13 C (University of Colorado Boulder, Boulder, CO, USA), with tetramethylsilane serving as the internal standard.Chemical shifts were reported in δ values (ppm).X-ray diffraction analyses were performed on a Bruker D8 VENTURE Super DUO diffractometer (Bruker, Billerica, MA, USA).

Procedure for the Synthesis of Compound 2
In a 100 mL round-bottom flask equipped with a magnetic stirrer, 1 g (4.901 mmol) of arylhimachalene and 1.42 g (17.5 mmol) of HBr were introduced.Then, 0.5 mL of H 2 O 2 was added dropwise over 1 h.The reaction mixture was stirred for 24 h at 0 • C. At the end of the reaction, monitored by thin-layer and gas chromatography, the mixture was neutralized by a saturated aqueous solution of NaHCO 3 , extracted with ethyl acetate (3 × 20 mL).The organic layers were gathered and dried over anhydrous magnesium sulfate (MgSO 4 ) and filtered.The solvent was removed in vacuo.The residue was purified by column chromatography using silica gel with hexane/AcOEt (98:2) as the eluent.Yield = 87%.Yellow oil.NMR

Procedure for the Synthesis of Compound 3
A mixture of arylhimachalene (1 g, 4.9 mmol) and 2 equivalents of N-chlorosuccinimide (NCS) in acetonitrile (10 mL) was stirred at 80 • C for 24 h.At the end of the reaction, monitored by thin-layer and gas chromatography, the mixture was extracted with dichloromethane (3 × 20 mL).Then, the organic layer was dried over anhydrous magnesium sulfate (MgSO 4 ) and filtered.The solvent was removed in vacuo.The residue was purified by column chromatography using silica gel with hexane/AcOEt (98:2) as the eluent.

Procedure for the Synthesis of Compounds 4 and 5
In a 100 mL three-necked round-bottom flask equipped with a magnetic stirrer, a dropping funnel, and a condenser, 1 g of (compound 2) or (compound 3) was introduced, followed by 4 mL of H 2 SO 4 .In an Erlenmeyer flask, a mixture of 1.2 mL of HNO 3 and 1.2 mL H 2 SO 4 was prepared and added to the dropping funnel.Then, the acid mixture was added dropwise to the reaction mixture within 1 h 30 min at 0 • C. The solution was then stirred for 12 h at room temperature.After completion of the reaction, 20 mL of a saturated NaHCO 3 solution was added to the reaction mixture, then extracted with ethyl acetate, washed with water, and dried over MgSO 4 .After the evaporation of the solvent, the crude product was purified by chromatography on silica gel with hexane/ethyl acetate (90:10) as the eluent.The recrystallization of the products was performed by slow evaporation from a hexane solution.

Crystal Structure Determination
Single-crystal X-ray diffraction measurements of the two synthesized products were carried out at room temperature using a Bruker D8 VENTURE Super DUO diffractometer [37] equipped with Cu-Kα radiation (λ = 1.5418Å) at 173 K (Table 6).The structure was solved by direct methods and successive Fourier difference synthesis using SHELXS 2014 [38] and refined by the full-matrix least-squares procedure on F2.The positions of non-hydrogen atoms were refined anistropically by SHELXL-2014 [39].Hydrogen atoms were placed in chemically acceptable positions.The ORTEP of the compound was generated using the ORTEP3 [40], and the packing diagram was generated using Mercury software [41].A total of 351 parameters were refined with 5862 unique reflections, which covered the residuals to R1 0.054.Crystallographic data were deposited in the Cambridge Crystallographic Data Centre as supplementary publications with CCDC numbers 2303321 and 2303322.Copies of the data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK by fax: 044 1223 336 033 or e-mail: deposit@ccdc.cam.ac.uk.

Computational Method
Full geometry optimizations of the arylhimachalene derivative substituted with bromine and chlorine were carried out using the Gaussian09 software package [43], employing the density functional theory (DFT) method.All calculations were performed utilizing the B3LYP hybrid density functional theory with the 6-31++G(d,p) basis set.Vibrational frequency calculations were conducted to verify that the optimized geometries represent local minima on the potential energy surfaces (PESs).The absence of imaginary frequencies confirmed that the stationary points corresponded to minima on the total PESs.
From the DFT calculation and after confirming the optimized structures, electronic properties related to the frontier molecular orbital energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were theoretically determined at the same level.Several local reactivity indices, such as chemical potential (µ), chemical hardness (η), electronegativity (χ), and electrophilicity index (ω), were cal-culated.These descriptors helped to understand the structure of the molecules and their reactivity.To gain further insights into the electrophilic and nucleophilic sites as well as hydrogen-bonding interactions in the synthesized arylhimachalene, the molecular electrostatic potential (MEP) was calculated.Additionally, the intermolecular interaction analysis of the crystal structure was determined through the Hirschfeld surface plots generated by using the Crystal Explorer [32].
3.7.In Silico Molecular Docking 3.7.1.Ligand Preparation Two halogenated nitro derivatives of arylhimachalene, namely bromo nitro-arylhimachalene and chloro-dinitro-arylhimachalene, which have been reported as dual inhibitors, were sketched using Maestro 12.8 (Schrodinger, LLC, New York, NY, USA) [44].A physostigmine inhibitor was employed as the standard (positive control) in this study.Each structure was assigned an appropriate bond order using the LigPrep package from Schrodinger [45].The inhibitors were then converted to SDF (Structures Data File) using Maestro and optimized using the optimized potentials for liquid simulations (OPLS 2005) force field with default settings [46].

Protein Structure Preparation
In this investigation, the structure of the Torpedo californica acetylcholinesterase complexed with UO2 (PDBID: 7B2W) was obtained from the RCSB protein data bank (RCSB PDB) [5].The PDB structure contains several instances of missing information regarding specific connectivity, bond orders, and formal charges.The protein structure was imported from the PDB into Maestro using the protein preparation wizard.Within this wizard, the option to display only polar hydrogen atoms was selected [47].This choice allows for the representation of solely polar hydrogen atoms in the structure [48].

Grid-Based Ligand Docking with Energetic
Grid-based lLigand dDocking with eEnergetic (GLIDE), a component of the Schrödinger suite, was utilized for docking analysis.It explores favorable interaction regions between ligands and proteins, employing a hierarchical series of filters to locate specific positions of the ligand within the protein's active site [49].GLIDE SP (standard precision) mode, designed for high-quality ligand poses, exclusively docks the top-scoring ligands.The docking process involves protein structure preparation, receptor grid generation, ligand structure preparation, and ligand docking.Subsequently, the chosen conformation is depicted in a 2D and 3D diagram, illustrating the ligand's interactions with active site residues using BIOVIA Discovery Studio 2021 [50].

Figure 1 .
Figure 1.Himachalene isomers, the major components of Atlas cedar oil.

Figure 2 .
Figure 2. Pharmaceuticals and natural products with benzocycloheptane motif.

Figure 1 .
Figure 1.Himachalene isomers, the major components of Atlas cedar oil.

Figure 1 .
Figure 1.Himachalene isomers, the major components of Atlas cedar oil.

Figure 2 .
Figure 2. Pharmaceuticals and natural products with benzocycloheptane motif.

Figure 2 .
Figure 2. Pharmaceuticals and natural products with benzocycloheptane motif.

Figure 4 .
Figure 4. Automatic fit of the two crystallographically independent molecules.

Figure 5 .
Figure 5. Dihedral angles between the planes formed by the nitro groups and benzene rings in the two independent molecules.

Figure 4 .
Figure 4. Automatic fit of the two crystallographically independent molecules.

Figure 4 .
Figure 4. Automatic fit of the two crystallographically independent molecules.

Figure 5 .
Figure 5. Dihedral angles between the planes formed by the nitro groups and benzene rings in the two independent molecules.

Figure 5 . 22 Figure 6 .
Figure 5. Dihedral angles between the planes formed by the nitro groups and benzene rings in the two independent molecules.Molecules 2024, 29, x FOR PEER REVIEW 6 of 22

Figure 10 .
Figure 10.Dihedral angles between the planes formed by the nitro groups and benzene ring.

Figure 10 .
Figure 10.Dihedral angles between the planes formed by the nitro groups and benzene ring.

Figure 10 .
Figure 10.Dihedral angles between the planes formed by the nitro groups and benzene ring.

Figure 12 .
Figure 12.Density distributions of the frontier molecular orbitals for the studied compounds, calculated using the B3LYP/6-31G++ (d,p) level of theory.

Figure 12 .
Figure 12.Density distributions of the frontier molecular orbitals for the studied compounds, calculated using the B3LYP/6-31G++ (d,p) level of theory.

Figure 15 .
Figure 15.Fingerprint plots of the major contacts of compounds 4 and 5.

Figure 14 .
Figure 14.Three-dimensional Hirshfeld surface of compounds 4 and 5 mapped with d norm .

Figure 15 .
Figure 15.Fingerprint plots of the major contacts of compounds 4 and 5.Figure 15.Fingerprint plots of the major contacts of compounds 4 and 5.

Figure 15 .
Figure 15.Fingerprint plots of the major contacts of compounds 4 and 5.Figure 15.Fingerprint plots of the major contacts of compounds 4 and 5.

Figure 16 .
Figure 16.The scale represents the number of conformations obtained for each energy level.

Figure 16 .
Figure 16.The scale represents the number of conformations obtained for each energy level.

Table 2 .
Theoretical and experimentally selected structural parameters of compounds 4 and 5.

Table 2 .
Theoretical and experimentally selected structural parameters of compounds 4 and 5.

Table 4 .
Energy (eV) and composition of some selected molecular orbitals of compounds 4 and 5.

Table 4 .
Energy (eV) and composition of some selected molecular orbitals of compounds 4 and 5.

Table 6 .
Experimental X-ray data collection from the synthesized products.