Coordination Chemistry, Antibacterial Screening, and In Silico ADME Study of Mononuclear Ni and Cu Complexes of Asymmetric Schiff Base of Streptomycin and Aniline

Two novel metal complexes, that is, Ni (StmAn)2(4) and Cu (StmAn)2(5), were synthesized from unsymmetrical Schi base ligand StmAn (3). e ligand was prepared by reuxing streptomycin (2) and aniline (1). ey were characterized by elemental microanalysis, conductivity measurements, and spectroscopic techniques such as H NMR, FT-IR, ESI-mass, and electronic absorption spectral study. Interestingly, the study revealed metal coordination through azomethine nitrogen and N-atom of NHCH3 of N-methyl-L-glucosamine unit of streptomycin.e electronic absorption spectral study supported an octahedral geometry for complex 4 and a tetrahedral geometry for complex 5. Particle size calculation by Scherrer’s formula indicated their nanocrystalline nature.e geometry optimization of the complexes was achieved by running anMM2 job in Gaussian supported Cs-ChemOce ultra-12.0.1 and ArgusLab 4.0.1 version software. Based on SwissADME predictions, a theoretical drug prole was generated by analyzing absorption, distribution, metabolism, excretion, and toxicity (ADMET) scores of the compounds. ey were screened for in vitro antibacterial activity study against four clinical pathogens such as E. coli, S. pneumoniae, P. vulgaris, and S. aureus. Minimum inhibitory concentration (MIC) study demonstrated greater inhibitory potency of complex (4) (0.024 g/L) for S. aureus relative to ligand (3) and complex (5). Studies show that metal complexes are more toxic to bacteria.


Introduction
e global community faces the crisis of e ective vaccines and medicines to treat current emerging diseases. A recent ongoing COVID-19 pandemic has destroyed the wellestablished human civilization in terms of several factors like social behavior, countries' economic status, and attitude to adapt to the existing environment [1]. It was a panic moment for everybody to come over from this disease. Many pharmaceutical organizations claimed their discovery of vaccines to control the current epidemic and also got partial success. Because of the genomic mutation process, pathogenic organisms change their biological functionalities and behave di erently [2]. It has become challenging to suppress their activity; instead, the scienti c community is trying to nd better alternatives to control their e ects [3,4]. e pharmaceutical industries run their drug discovery programs to come out of the pandemics and save human lives. A similar problem that medical science faces is the antibiotic crisis and unethical use of antibiotics. Even most antibiotic drugs stored in the drug library have undergone antibacterial resistance, and currently, there are no new antibiotics to ght such diseases [5][6][7]. e discovery of antibiotics in 1930 was a great invention of medical science. During the third and fourth quartiles of the 20 th century, antibiotics were on top, especially for treating injured people of post-World War II [8,9]. e current antibiotic research and development have become less innovative as the pharmaceutical industry has failed to introduce a new class of antibiotics [10]. Most of the present antibiotics were discovered in the 1940-1960s. At that time, the disease treatment procedure depended on antibiotics, and even today, its use has not been denied [11].
Recently, antibiotics have become the most successful chemotherapy drugs in the history of modern medicine and are therefore considered a miracle drug [12]. e current antibiotic research has been considered the most challenging and exciting field of pharmacology due to the increasing threat of drug resistance, which has sprouted the need for fresh ideas and strategies to develop it anew [13]. Mismanagement in the use of antibiotics, a high rate of antibacterial prescription, and large-scale antibiotic use in clinical practice are the major causes of drug resistance [14]. erefore, to address the severe challenges of multidrug resistance, the frequency of antibiotic research should necessarily be enhanced to gear up novel antibiotics' discovery. We have selected Schiff base compounds and their metal complexes as role models for antibiotics trial discovery to check potential drug activities. e Schiff bases are an important class of organic compounds and are known for their drug activities. Recently, coordination chemistry of Schiff bases is gaining much popularity due to their versatile applications as potential drug candidates to ensure better and more successful interaction with the disease-causing organisms. Like a drug, they have potential applications as an antibiotic [15,16], antitumor [17], antimicrobial, antiviral [18], and anti-inflammatory [19]. e Schiff bases containing azomethine (-C � N-) linkage as the integral part of several patented modern drugs have enamored pharmaceutical research activity and directed the path for the discovery of new chemotherapeutics [20]. e nucleophilic nature of azomethine nitrogen can donate electrons to the electrophilic substrate due to Lewis base nature and consequently participates in chelation chemistry. e stable chelate complex forms by coordinating metal ions with electrondonating Schiff base molecules [21]. Besides the drug activities, Schiff base transition metal complexes have wide applications in many other fields such as industries, organic and inorganic synthesis as the catalyst, polymer chemistry, and many more [22,23]. e present research attempts to change the structural and physiological profile of streptomycin by changing ligand property by forming a Schiff base and its complexation with metal ions.
Most aminoglycosides as antibiotics are toxic, and their use is a question of great human concern in clinical science. Streptomycin ( Figure 1) is a "first-generation aminoglycoside," typically used in the treatment of highly contagious disease, "Tuberculosis," and exerts the bactericidal effect by inhibiting ribosomal protein synthesis [24]. e most crucial side effects of streptomycin are ototoxicity and nephrotoxicity, which restrict its usefulness, and modifying its structural design is necessary to reduce its toxicity [25]. A significant portion of its structure is the streptose ring with the -CHO group that plays a vital role in the formation of Schiff base by the condensation with -NH 2 group of aniline. e metal ion is nested in the Werner type compartment and stabilizes the complex molecule due to rich redox chemistry [26]. Since there are several published articles on metal complexes of Schiff bases, we focus our research on streptomycin-based Schiff base ligand. e present investigation reports the synthesis, spectral characterization, coordination behavior, X-ray powder diffraction study, and antibacterial evaluation of Ni II and Cu II complexes of Schiff base ligand obtained from the condensation reaction of free streptomycin and aniline. e work has been extended to evaluate drug profile of the synthesized compounds by generating ADMET scores through SwissADME prediction web server.

Experimental Section
2.1. Materials. All the chemicals used were of analytical reagent grade (AR) type, available commercially. Streptomycin sulphate (Alfa Aesar) and aniline (Merck) with highgrade purity were used as starting materials to prepare ligand 3.
e solvents and reagents used for synthetic purposes were procured from commercial sources and purified by standard procedures. e metal salts were CuCl 2 .2H 2 O and NiCl 2 .6H 2 O (Merck). Distilled methanol (Qualigen) was used as the solvent for the synthesis. e bacterial culture was prepared in Muller Hinton's agar media (Himedia co.). All the glassware used in the research process was of highgrade borosilicate glass, and they were cleaned with triple distilled water before the research operation.

Instruments.
e melting point was recorded on an OMEGA melting point apparatus.
e elemental microanalysis was performed on the Elemental Vario EL III Germany model analyzer. e pH measurement was done in the Elico-16 pH meter. FT-IR spectra were recorded on a Perkin Elmer 783 FT-IR Spectrophotometer on KBr discs at the range of 4000-400 cm −1 . Electronic absorption spectra were recorded on a single beam microprocessor UV/Vis. Spectrophotometer (LT-290 model, India) used DMSO as the solvent from the 200-800 nm range. 1 H NMR spectrum of the ligand was recorded on a Bruker AV300/1 FT-NMR spectrometer using DMSO-d6 as solvent and TMS as an internal reference. ESI-mass spectra were recorded as TOF-MS on Agilent 6520 Q-TOF mass spectrometer equipped with an electron spray ionization source. e XRD powder pattern was recorded on a vertical type Bruker AXS d8 Advance X-ray diffractometer with Cu-Kα line (λ = 1.54056Å) as the radiation source under slow-scan over the range of 5°-80°as a function of (2θ). Crystallographic data were analyzed on X'Pert High Score and Origin software programs. e metal complexes' possible geometries were evaluated using the molecular modeling calculation, optimized by the CsChem 3D Ultra-12.0.1 program and Argus Lab 4.0.1 version software.  O 11 ). To the solution of Stm (2), (2 mmol, 1.359 g) in 30 ml hot aqueous methanol was added 2 mmol (0.2 ml) of ligand precursor (1), and the pH of the solution was adjusted to slightly alkaline (pH 8) by adding 2N NaOH solution dropwise. e mixture was left under reflux for about 3 h, and the volume of the solution was reduced by half when the pink-colored solid product of StmAn (3) was separated by a slow diffusion process, purified by recrystallization from hot methanol, and dried in a desiccator. e synthetic route for the preparation of ligand (3) and complexes (4) and (5)

Antibacterial Activity Study.
e ligand (3) and its metal complexes (4) and (5) were screened in vitro for their antibacterial activity against clinical strains of two Grampositive (S. aureus and S. pneumoniae) and two Gramnegative (E. coli and P. vulgaris) bacterial pathogens. e standard Kirby-Bauer paper disc diffusion method was followed to determine the antibacterial potency. e test solutions were prepared in DMSO at three concentrations (100, 50, and 25 μg/μL). e well-sterilized filter paper discs of 5 mm diameter (Whatman No. 1) were impregnated with test compounds at their specific concentrations and carefully stuck on the previously seeded bacterial culture in Petri plates [27,28]. Amikacin 30 μg/disc of 6 mm size (HIMEDIA co.) was used as standard (+ve control). e antibacterial activity of the test compounds was also compared with the parent drug streptomycin. Afterward, Petri plates were incubated at 37°C, and the diameter of the zone of inhibition around each disc was measured after 24 h of incubation with the help of an antibiogram zone measuring scale. Since the synthesized compounds were sensitive to all selected pathogens in the disc diffusion study, they were further assessed for MIC studies. MIC stands for the minimum concentration of the test compounds, which inhibits bacterial growth. For this study, stock solutions of the test compounds at 50 μg/μL concentrations were prepared in DMSO and followed twofold broth microdilution method to prepare their lower concentration solutions. e target organisms were cultured in nutrient broth incubated at 37°C for 24 h. To 2 mL of each diluted test solution prepared according to broth microdilution method, 0.1 mL bacterial suspension was added, and they were incubated at 37°C for 24 h to observe bacterial growth. e turbid solution represents bacterial growth. e stock solution mixed with bacterial suspension was referenced as a positive control, and the other containing broth loaded with bacterial suspension was referenced as a negative control. e tests were done in triplicate to reduce error.

Physical Characterization.
Schiff base ligand and its metal complexes were analyzed using CHN analysis to validate their molecular formulae. e CHN and other physical properties data of the compounds, including molar conductance, pH, and color, are given in synthesis section. Based on the analytical results, the complexes exhibit a 1 : 2 metal-ligand ratio and a ML 2 type stoichiometry. e molar conductance values ranged from 7.41 to 31.8 μSiemen/cm, which indicates their nonelectrolytic nature, and these values are smaller than expected for an electrolyte [29]. e crystals of complex 4 are hygroscopic, which indicates that water molecules are involved in crystal formation. Metal complexes synthesized from the ligand undergo a change in color, indicating that they are the product of chemical reactions [30,31]. Dropping pH values can also promote complexation with metal ions due to the deprotonation of ligands. In our work, pH values decrease from ligand to metal complexes, confirming complexation.  [32]. is shift of IR band positions indicates the coordination of the azomethine nitrogen with the metal centers. A sharp band around 1100 cm −1 for all the compounds is attributed to the ] (C-N) stretch of the N-methyl group attached to the N-methyl-Lglucosamine ring of Streptomycin moiety [33]. Medium intensity bands evidence the metal-nitrogen bonding in metal complexes at 457.09 and 465.71 cm −1 characteristics for complexes 4 and 5, respectively [34]. e metal-oxygen bonding in complex 4 is substantiated by forming a medium intensity band at 505.80 cm −1 [35].

Spectroscopic
us, the IR spectral results provide strong evidence for the complexation with Schiff base and suggest that the ligation is likely due to azomethine nitrogen and the nitrogen atom of the CH 3 substituted amine group in the N-methyl-L-glucosamine component of Schiff base. ese data indicate that coordination to the metal centers occurs via the N,N donor atoms of ligand 3.

1 H NMR Study.
e 1 H NMR spectrum of ligand 3 is shown in Figure S4, and the spectral data are summarized in Table 1. e integral intensities of each signal in the spectrum agree with the number of different types of protons present. e 1 H NMR signal around 2.5 ppm is assignable to DMSO solvent protons. e signal at 1.21 ppm is suggested to be the peak for CH 3 protons attached to the tetrahydrofuran ring. e azomethine proton appears as a singlet at 7.983 ppm [36]. All the four aromatic protons as a set of two doublets appear in the range of 6.956-7.4 ppm. Other protons of the tetrahydrofuran and tetrahydropyran ring show peaks in the region of 3.798-4.983 ppm. e peak in the region between 2.67-2.71 ppm is assignable to N-CH 3 protons [37]. All these results are in good agreement with the proposed molecular formula of the ligand.

Electronic Absorption Spectral Study.
e electronic absorption spectra of the synthesized compounds 3, 4, and 5 in DMSO were studied in the range of 250-800 nm at room temperature [38]. e spectral comparison of the free ligand with its metal complexes showed the persistence of the bands representing n ⟶ π * and π ⟶ π * transitions for the ligand in all the complexes. However, the bands have undergone a bathochromic shift in the regions of the visible spectrum in all the complexes [39]. e new bands were also observed in the spectra of metal complexes. e electronic absorption spectra provide valuable information about possible geometry of the metal complexes, and ligand arrangement. e absorptions below 400 nm are attributed to transitions within the ligand orbital and ligand-metal charge transfer, whereas those in the visible region are due to d-d transitions. e electronic absorption spectrum of the ligand shows a peak at the wavelength of 319 nm assignable for π ⟶ π * transitions. e broad closely spaced peaks at 340-347 nm are assignable for the intraligand n ⟶ π * transitions. e electronic absorption spectrum of complex 4 displayed three d-d transition bands at 761 nm : 3 A 2g (F) ⟶ 3 T 2 g (F), 696 nm: 3 A 2g (F) ⟶ 3 T 1 g (F), and 398 nm: 3 A 2g (F) ⟶ 3 T 1 g (P), supporting its octahedral geometry. e high energy bands at 305-320 nm range are attributed to π ⟶ π * transitions for azomethine and aromatic π electrons [40,41]. e electronic absorption spectrum of the complex 5 displayed only one d-d transition band at 650 nm corresponding to 2 T 2g ⟶ 2 e g transition, which supports its tetrahedral geometry [42,43]. e electronic absorption spectra of all the three synthesized compounds are shown in Figure 2.

ESI-Mass Study.
e molecular ion peaks for the compounds have been considered to determine the proposed molecular weight. A positive ion ESI-mass spectrum of the ligand (3) showed a peak at m/z 656.68 amu corresponding to molecular ion peak [M_ + ], which confirms the proposed molecular formula of the ligand. e series of peaks in the range m/z 642. 7

PXRD Study.
e powder X-ray diffraction technique was applied to get different crystal information [44,45]. Powder diffraction patterns of the ligand and metal complexes ( Figure 4) revealed their crystalline nature. Crystal data and structure refinement analysis data are also summarized in Table 2. e average crystallite size of the ligand (3) and its metal complexes (4) and (5) was calculated from Scherer's formula, d XRD � 0.9λ/βCosθ, where λ is the wavelength, β is the full-width at half maximum of the characteristic peak, and θ is the diffraction angle for the reflection plane [46,47]. Eleven reflection peaks had been recorded for the ligand (3) with maximum intensity at 2θ � 44.64°and interplanar distance d � 2.028Å. For the complex (4), 12 reflection peaks had been observed with peak maxima at 44.65°and a corresponding d spacing value of 2.03Å. Complex (5) exhibited 11 reflection peaks with an intense peak at 15.45°with a corresponding d spacing value of 5.73Å. e average crystalline size was calculated as 10.81 nm, 12.72 nm, and 11.96 nm for the ligand (3), and complexes (4) and (5) respectively, revealing their nanocrystalline nature.
To find the number of dislocation lines per unit area (nm −2 ) of ligand (3) and its complexes (4) and (5), the equation δ = 1/α 2 was used, in which α represents the average crystallite size [48]. It is one of the factors that cause crystallographic defects. e dislocation density of 3 varied from 2.67 × 10 −3 to 148.22 × 10 −3 nm −2 , which is different from the dislocation density of complex (4) (δ = 3.61 × 10 −3 to 12.80 × 10 −3 ) nm −2 and (5) (δ = 3.53 × 10 −3 to 18.69 × 10 − 3 ) nm −2 . Different dislocation densities of ligand relative to complexes have proven the formation of complexes by the interaction of the ligand with metals. Additionally, the variation in their lattice parameters was also evidenced by the comparative study of microstrain (ε). Here, ε of the ligand and its metal complexes were calculated by the equation ε = β/4tanθ, where β stands for FWHM, and θ represents the diffraction angle [49]. e microstrain of ligand and its metal complexes are shown in Table 2. Microstrain is another factor that leads to crystallographic defects.
e ε values are in the range of

Molecular Modeling Study.
To better understand hexa-and tetra-coordination of complexes (4) and (5), 3D molecular modeling of the proposed structure was carried   [50,51]. e study revealed octahedral geometry for complex (4) and tetrahedral geometry for complex (5). e correct stereochemistry was established by modifying molecular coordinates to obtain low energy and suitable molecular geometry [52]. e most stable conformer was fully optimized with molecular orbital functions PM3 supported in ArgusLab molecular modeling software [41]. Several attempts at energy optimization were made to determine the minimum. Some selected bond lengths and bond angles are listed in Table 3, and the optimized structures with atom labeling of the complexes are presented in Figures 5(a) and 5(b). e modeling software shows that the bond angles around the metal centers strongly support octahedral and tetrahedral geometries for complexes (4) and (5), respectively. e minimum geometrical energy of the complexes, optimized by MM2 calculation in CsChemOffice 3D Ultra software, was similar to the calculations made in ArgusLab software. e final geometrical energy for complexes (4) and (5) was 493.4026 and 991.4023 Kcal/mol. Furthermore, the change in bond lengths between metal-nitrogen and the metaloxygen complexes compared with the ligands suggests coordination. e azomethine C�N bond length of the ligand (1.265Å) has been increased to (1.286Å/1.281Å) and (1.270Å/1.291Å) for complexes (4) and (5), respectively.

Antibacterial Activity Study.
Recent advances in pharmaceutical research have stressed the need for metals to be used in drug delivery systems as chemotherapeutic agents [53]. Metals provide templates for synthesis, but they also introduce functionalities that enhance drug delivery vectors [54]. Metals are biologically inert, and many organic drugs require interactions with metals for optimal activity [55,56]. erefore, attempts were made to explore the antimicrobial activity of ligand (3), and its complexes (4) and (5).
In this work, four clinical isolates of bacterial pathogens viz. E. coli, S. pneumoniae, P. vulgaris, and S. aureus were selected for in vitro interaction with the ligand 3 and its metal complexes (4 and 5). Antibacterial activity was evaluated by measuring the diameter of the zone of inhibition around each disc in mm. e antibacterial results presented in Table 4 showed that complexes 4 and 5 are relatively more active than the ligand 3 and its parent compound (Streptomycin) against all the four bacterial pathogens.
e comparative antibacterial activity of the synthesized compounds and control drug Amikacin with all the bacterial pathogens is shown in the bar graph in Figures 6-8. e antibacterial data demonstrate the more excellent antibacterial activity of the synthesized compounds at their higher concentration. Moreover, the antibacterial growth inhibition activity of the complexes was enhanced after the chelation of metal with the ligand. eir MIC studies demonstrated a precise measurement of the antibacterial activity of the ligand and complexes. e lowest MIC value (0.048 μg/μL) was reported for ligand 3 with S. aureus bacteria followed by S. pneumoniae (0.097 μg/μL). Complex 4 has shown the lowest MIC value (0.024 μg/μL) with S. aureus bacteria. Likewise, complex 5 has also shown greater antibacterial potency with S. aureus and S. pneumoniae organisms. Overall, the MIC study revealed more significant antibacterial activity of all the synthesized compounds with Gram-positive bacteria. e studied metal salts have shown no remarkable antibacterial potency in MIC values. With S. aureus bacteria, complexes (4) and (5) have shown two multiples more active than 3 and four multiples more active than compound (2). Similarly, the MIC study also revealed more excellent activity of complexes relative to compounds (2) and (3) with S. pneumoniae bacteria. Concerning E. coli, complex (4) has shown more excellent antibacterial activity than other compounds. e MIC data are presented in Table 5. e enhanced antibacterial activity of the complexes concerning the ligand can be explained based on Tweedy's chelation theory. Chelation reduces the polarity of the metal atom by sharing charge with donor atoms in the heterocyclic ring and provides stability of the complex. Delocalization of charge over the whole chelate ring enhances the complex's lipophilic character and increases penetrating power into the lipid membrane. Finally, the growth of microorganisms is deactivated by blocking protein binding sites in the enzyme of microorganisms. e antibacterial toxicity of the complexes is higher for Grampositive strain relative to the Gram-negative strain of the bacterial pathogens. e Gram-negative strain bacteria have a hydrophilic polysaccharide chain in the outer cell membrane, disturbing the free penetration of complexes and showing relatively low toxicity.

Swiss ADME Study.
Medicinal chemistry is one of the fastest-growing fields of today's research. On the dark side, it takes more than ten years and billions of money to develop a drug [57]. In fact, from several thousand to millions of compounds, one compound may pass all the criteria to become an approved drug. Over 50% of these compounds fail the physicochemical properties tests, and the drug development processes are greatly helped by assessments like ADMET properties, which aim to predict the hit compounds (Table 6) [58].
Absorption, distribution, metabolism, excretion, and toxicology, shortly referred to as ADMET, are associated with the fate of a chemical compound in our body. Swiss ADME is a freely available web server that computes the physicochemical properties based on the structure of the compound (http://www.swissadme.ch/). Because of having large structures, the metal complexes could not be computed. So, the in silico properties of the prepared Schiff base are compared with those of one of its precursors, streptomycin. It is interesting to note that the streptomycin drug, which has been approved as a drug, was found to possess such intriguing physicochemical properties, many of which do not "fit" the suitable criteria range as stated here [59] and otherwise [58]. e mcule web server (https://mcule.com/), which along with other properties, is equipped to evaluate the toxicity of the compound presented.
is mcule web server reported a potential toxic substructure found for the Schiff base and the precursor antibiotic streptomycin. e data of pharmacokinetic and drug-likeness results are reported in Tables S1 and S2. Furthermore, the compound showed higher synthetic ability and lower bioavailability scores. Due to "failed" physiochemical properties, both compounds failed in almost all the drug-likeness and medicinal chemistry filters the Table 3: Selected bond lengths, bond angles, and energy parameters of metal complexes.

Conclusion
Two new metal complexes (4) and (5) were successfully synthesized from aminoglycosidic Schiff base ligand (3), obtained by condensation of starting compounds 1 and 2.
Besides the polydentate nature of 3, its complexation with Ni (II) and Cu (II) ions occurred through N, N donor atoms.
Various spectral studies validated this study. All compounds synthesized were nonelectrolytic based on the molar conductance data. e IR spectral study confirmed the ligandmetal coordination through azomethine nitrogen and N-atom of N-methyl-L-glucosamine ring of Streptomycin moiety, which was evidenced by shifting of the peak values in the spectra of complexes. e particle size calculation by Scherrer's formula indicated their nanocrystalline nature. An octahedral geometry for complex (4) and a tetrahedral geometry for complex (5) have been assigned, which were further supported by various molecular modeling MM2 calculations and energy optimizations. e antibacterial data revealed better growth inhibition effect of ligand (3) relative to starting drug compounds (2) and control drug Amikacin. e metal complexes exhibited enhanced antibacterial activity against all bacterial pathogens. Data from SwissADME and Mcule web server reported potentially toxic substructures for ligand (3) and precursor antibiotic streptomycin.

Data Availability
All the data are included in the manuscript and are available for the readers.

Conflicts of Interest
e authors declare that they have no conflicts of interest.