Crystal structure, spectral investigations, DFT and antimicrobial activity of brucinium benzilate (BBA)

The unreported brucinium benzilate (BBA) crystal and Hirshfeld surface analysis indicated the influence of intramolecular hydrogen bonding network on the crystal structure. Protonation occurs at the tertiary nitrogen as it is the most basic site. The protonated N-H+ proton was observed at 7.08 ppm and the benzilate carbon COO- at 178.41 ppm. Molecular electrostatic potential (MEP) studies showed the electron-rich and electron-deficient sites in the molecule for understanding BBA interaction with an enzyme. Frontier molecular orbital (FMO) studies indicated that BBA molecule is thermodynamically stable and the HOMO-LUMO energy gap was found to be 4.454 eV. The highest interaction has the energy (322.86 kcal/mol) between tertiary ammonium N(LP) and H+. Inhibition tests showed that brucinium benzilate inhibits Bacillus cereus and Salmonella typhimurium bacteria. ADMET properties indicated that BBA has drug characteristics in binding plasma protein.

Recently, benzilic acid derivatives were recognized as a potent antimicrobial agent with good activity range [18]. Benzilic acid shows multi-use such as polymers, medicine, and analytical agent [19]. Brucine and benzilic acid exhibit a variety of medicinal uses. In the present study, the synthesized brucinium benzilate compound was characterized by single crystal XRD, ATR-IR, FT-Raman 1 H NMR, and 13 C NMR analysis. DFT calculations were performed using the crystallography information and the theoretical results were compared with the vibrational experimental results. Computational analyses like HOMO-LUMO, natural bond orbital (NBO) and molecular electrostatic potential (MEP) analysis were performed to know about molecular properties. ADMET properties of the material are reported to throw light on its biomedical applications.

Synthesis of brucinium benzilate
Brucine (AR, LOBA) and benzilic acid (AR, Merck) were taken in equimolar ratio to prepare brucinium benzilate solution in ethanol. The solution was filtered using Whatman filter paper twice and the filtrate was allowed to crystallize. Repeated recrystallization in ethanol yielded the quality crystals after 24 days.
Single crystal X-ray diffraction The structure determination (CCDC NO: 1966353) was done by the direct method using SHELXT-2014/4 [20] and refined using full-matrix least-squares on F 2 method using SHELXL2018/3 [21]. Multi-scan SADABS [22] was used to perform area-detector scaling and absorption corrections. Anisotropic displacement parameters were assigned to locate non-hydrogen atoms.

Spectroscopy
ATR studies were done using Perkin Elmer Spectrum one. FT-IR Spectrometer. The FT Raman spectrum was recorded in the spectral range of 4000-50 cm -1 using Bruker RFS 27 FT Raman spectrometer. NMR studies were done using the AVANCE III 500 NMR spectrometer.

Computational details
Quantum chemical calculations were performed on the title molecule by applying DFT method using the Gaussian 09 program suite [23] at the Becke-3-Lee-Yang-Par (B3LYP) level [24,25] combined with the standard 6-31G (d,p) basis set and M06-2X functional at the basis set 6-31G (d,p) respectively. During the optimization procedure, all parameters were allowed to relax to obtain a stable structure with minimum energy. The minimum global energy of the title compound was ascertained from the structure optimization procedure. The natural bond orbital (NBO) analysis was performed using NBO 5.0 program [26] as implemented in the Gaussian 09 package at DFT/B3LYP level. The hyperconjugation and the interaction energy within the molecule were deduced from the second-order perturbation approach [27][28][29].

Results and discussion
Crystallographic and structural details BBA crystallized (Table 1) in an ionic form by forming protonated brucinium cation and deprotonated benzilate anion (Fig. 1). Brucinium cation consists of seven rings in the structure with one aromatic ring consisting of two methoxy groups. The O5 and O7 methoxy groups formed a torsional angle C

Optimized structure
Structure optimization is performed on BBA crystal structure to calculate theoretically the most stable geometry, bond lengths and bond angles using DFT/B3LYP at 6-31G(d,p) level and M06-2X functional at the basis set 6-31G(d,p). The selected bond lengths and bond angle values were compared with XRD data in (Table S1). Small variations were observed between the XRD and its optimized geometry because DFT calculations assume the interactions in a gaseous

Hirshfeld surface analysis
The Hirshfeld surface analysis is an essential tool for understanding the interactions [30]. The intermolecular interactions were analyzed by crystal explorer 3.1 [31], which produce

Frontier molecular orbital (FMOs)
Frontier molecular orbital (FMOs) plays a significant role in electrical and optical properties and are also used in the assessment of the chemical properties of the molecule [32]. The molecular orbital functions are plotted as surfaces around the molecular structure. The HOMO exhibits the ability to donate electrons and LUMO exhibits the ability to accept electrons. The HOMO and LUMO energy gap are the most important parameters for chemical reactivity and kinetic stability of the molecule. A large value of the HOMO-LUMO energy gap means high kinetic stability and low chemical reactivity. The lower HOMO-LUMO energy gap is the most significant parameter for the chemical reactivity, which explains intramolecular charge transfer (ICT) within the molecules [33] which is responsible for the bioactivity of the molecule.  (Fig. 6). The HOMO is located on C 29 , C 37 , C 38  In brucine the methoxy groups attached to benzene ring will increase the electron charge density. The benzilate aromatic rings also have higher electron charge density. Therefore, no charge transfer can occur between these aromatic rings. So, HOMO-LUMO gap is higher. From the HOMO and LUMO energy, and the density functional theory (DFT) were used to realize the chemical reactivity. The electron affinity (A) and ionization potential (I) are equal to orbital energies of HOMO-LUMO as A= -E LUMO and I = -E HOMO. The electron affinity and ionization potential were found as 5.7006 and 1.2466 eV, respectively. The electronegativity (χ= IþA 2 ), chemical potential (μ = -χ), chemical hardness (η = ΔE 2 ) chemical softness (σ = 1 2η ), electrophilicity index (ω = μ 2 2n ) and nucleophilicity index (N = 1 ω ) are linked to their chemical reactivity and electrostatic surface. Electrophiles are classified based on the electrophilicity index as marginal (ω <0.80eV) moderate (1.50 > ω>0.86 eV) and acid strong (ω>1.50eV). The nucleophiles are classified as moderate (3.00 N >2.00eV), marginal (N<2.00 eV) and strong (N>3.00 eV) [34]. According to the classification, the compound is a strong electrophile and marginal nucleophile. The strong electrophilic nature of the title compound suggests that in a biological activity it can accept electron density.

Stabilization energy of natural bond orbital (NBO)
The natural bond orbital calculation was carried on the compound to determine the electron donor-acceptor interactions in the molecule using NBO 5.1 programmer implemented in the Gaussian 09W package at the DFT/B3LYP/6-31G(d,p) level of theory. The orbital prefers the hyper conjugative and resonance. The highest interaction energy was between the N79-LP(1)→H84-LP*(1) was 322.86 kcal/mol; this is the interaction between the most basic nitrogen in brucine with a proton. The next highest energy interaction was between O27-LP(2)→H84-LP*(1) (118.82 kcal/mol). This interaction is due to -COOand H +, which is expected to be stronger. The interaction energy of 74.80 kcal/mol was due to carboxylate    Fig. 11 Experimental and theoretical Raman spectra anion resonance energy which is a stable one. Other minor energy interactions like π→π*, σ→σ*, n→π*, and n→σ* have also been computed and their values were found to be lesser (Table S2). The conjugation between N-C=O was calculated as 51.53 kcal/mol.

Molecular electrostatic potential (MEP)
The electric charge distribution in the molecule influences the following factors such as vibrational spectroscopy, electrostatic potential, and acid-base properties [35]. The molecular electrostatic potential (MEP) is an effective tool for identifying and ranking the hydrogen bond donating and accepting sites in organic compounds [36]. MEP correlates crystal packing with the relative strengths of hydrogen-bond donors and acceptors for understanding intermolecular interactions and other properties of crystalline materials [37]. The molecular electrostatic potential image (Fig. 7) of brucinium benzilate [BBA]. Red and yellow regions indicate the high electronegative potential on benzilate moiety due to C=O carbonyl group. From molecular electrostatic potential, it is easy to identify electron-rich and deficient sites. The red and yellow regions on benzilate anion is an electron-rich site and most favourable for the electrophilic attack. The blue colour region in brucinium cation indicates electron-deficient site which favors nucleophilic attack. The green region in the BBA molecule indicates the neutral site. The drug-receptor and enzymesubstrate interactions of the molecule can be predicted by molecular electrostatic potential map. 1 H and 13 C NMR spectroscopy δ in ppm; 7.77 -7.52: (Aromatic H); 7.322 -7.24 (Aromatic H) [38]; 7.00: (Tertiary amine NH + ) (Fig. 8). δ in ppm; 178.41: (COO -) [39,40] (Fig. 9).

Vibrational frequencies analysis
The vibrational frequencies of the BBA molecule were calculated theoretically using DFT/B3LYP at 6-31G (d,p) level and M06-2X functional at the basis set 6-31G(d,p). The IR and Raman spectra of BBA are presented in Figs. 10 and 11, respectively and their corresponding peak's assignments are tabulated in Table S3.  [41,42] have been noticed.

Biocidal activity
The antibacterial activities of synthetic compounds were evaluated by the disc diffusion method. It was identified by  (Table 3) and antibacterial activity photograph is shown in Fig. 12. It was found that the bacterial strain Salmonella typhimurium exhibits the highest inhibition zone (24 mm, 15 mm, and 11 mm) refers to the diameter of the halo in all the three concentrations (1000μg/ml, 750μg/ml, 500μg/ml) of BBA where the activity increases with increase in concentration. Results reveal that for bacterial strains, activity increases with an increase in compound concentration and the inhibition of the strains also increases. Bacillus cereus and Salmonella typhimurium used in this study and compared with standard Ampicillin.

ADMET properties
ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) profile of title compound and the standard drug reference of Regorafenib were evaluated using PreADMET [43] webserver. Caco-2 permeability and blood-brain barrier crossing ability of BBA were higher than the Regorafenib. Human intestinal absorption (HIA) property of the title compound was examined using drug filter rules which confirmed that the proposed drug had little higher HIA property than standard drug. The efficiency depends on the quantity of plasma protein binding (PPB) ability. The compound had higher efficiency than that of Regorafenib. The compound obeys Lipinski ' s five rules (molecular weight <500, not more than five hydrogen bond donors, not more than ten hydrogen bond acceptors and a partition coefficient (log P) value <5). The insilico synthesized compound possesses good drug-likeness as well as ADMET properties [44] (Table 4).

Conclusion
The Hirshfeld surface analysis and its associated 2D fingerprint plots have been used for a detailed exploration of molecular interactions.