Synthesis, Characterization, and Antibacterial Evaluation of Heteroleptic Oxytetracycline-Salicylaldehyde Complexes

A new series of mixed ligand complexes of Cd(II) and Mo(V) were successfully synthesized by refluxing the mixture solution of oxytetracycline hydrochloride (OTC.HCl) with an aqueous and alcoholic solution of metal (M�Cd(II) and Mo(V)) salts and an alcoholic solution of salicylaldehyde (Sal). +e complexes were characterized by modern analytical and spectral methods such as elemental microanalysis, pH, conductivity, surface tension, viscosity, melting point, and spectral methods such as FT-IR, NMR, electronic absorption, SEM, and mass spectrometry. Conductivity measurements of the complexes revealed their electrolytic nature.+e kinetic and thermal stabilities were investigated using thermogravimetric and differential thermal analysis techniques. +ermodynamic and kinetic parameters such as E∗, ΔH∗, ΔS∗, and ΔG∗ were calculated from TG curves using the Coats–Redfern method. Geometry optimization of the proposed structure of the complexes was achieved by running MM2 calculations in a Gaussian-supported CS ChemOffice 3D Pro.12.0 version software. +e final optimized geometrical energies for respective CdOTC/Sal and Mo-OTC/Sal complexes were found to be 923.1740 and 899.3184 kcal/mol. +e electronic absorption spectral study revealed a tetrahedral geometry for the Cd-OTC/Sal complex and octahedral geometry for the Mo-OTC/Sal complex. +e antibacterial sensitivity of the complexes was evaluated against three bacterial pathogens such as S. aureus, E. coli, and P.mirabilis using the modified Kirby–Bauer paper disc diffusionmethod.+e antibacterial study revealed significant growth inhibitory action of the complexes.


Introduction
e current interest in the improvement of the functionality and applicability of metal complexes has become an important part of coordination chemistry research [1,2]. Coordination compounds containing metals bound in the mesh of ligands have a variety of biological functions because many of them are used in the treatment of diseases in medical science [3]. Besides their biological functions, they are also used in chemical sciences as catalysts [4], reaction templates, and reaction activators [5]. e chief process for the fabrication of metal complexes is redox chemistry, where the metals provide a vacant orbital to the ligand for chelation.
e ligand functions as an electron donor and behaves as a Lewis base. e formation of the complex is therefore a simple Lewis acid-base reaction [6]. We have focused our research intended to address the antibacterial significance of the metal complexes of the oxytetracyclinesalicylaldehyde mixed ligand. Recently, antibiotic discovery has become a critical issue in pharmaceutical science due to the increased risk of drug resistance [7,8]. Medical science has established antibiotic therapy for the treatment of bacterial pathogens, and antibiotics are now considered one of the core drugs of modern medicine. Over the past few decades, these drugs have been losing their foundation in fighting bacteria. e bacterial strains are changing genomic characters by mutation process, and they are no longer affected by used antibiotics that once suppress their growth and activity [9]. Several natural antibiotics have lost their activity, and we need to synthesize laboratory-based antibiotics to overcome the antibiotic resistance crisis. erefore, it can be considered a better option to control disease outbreaks by metal-based drugs in an effective way [10,11]. In this research study, the ligands used for heteroleptic complex formation are oxytetracycline and salicylaldehyde.
Oxytetracycline is a broad-spectrum antibiotic of the class tetracycline that performs antibiotic functions by inhibiting protein synthesis in bacteria. It was first isolated from the soil bacteria Streptomyces rimosus in 1948, patented in 1949, and came into commercial use in 1950 [12]. It stands parallel in properties to tetracycline, which is commonly used as a veterinary antibiotic. In humans, it is used to treat eye infection trachoma, genital infection, urethritis, chest infection, psittacosis, and pneumonia. As a veterinary medicine, oxytetracycline is used for the treatment of infections in animal husbandry and fish farming [13,14], in agriculture as a pesticide, and as a dietary supplement for livestock [15]. In the past few decades, its use as an antibiotic has become less common due to increased antibacterial resistance among targeted pathogens. Structurally, oxytetracycline ( Figure 1) has a naphthacene ring skeleton similar to tetracycline, with many chromophoric groups responsible for metal attachment in complex formation. It is a type II bacterial aromatic polyketide containing one aromatic ring.
ere is a structural difference between tetracycline and the presence of a substituent group -OH at the C5 of ring B [16].
is makes a vast difference in the physiological and biological profiles of the compounds. In a survey of its toxic profile, oxytetracycline can complex with Ca and Mg in vivo in the human body and cause severe physiological defects [17,18]. Besides the in vivo physiological defects, it can also eliminate toxic heavy metals from the human body by complex formation processes. Almost 50-80% oxytetracycline undergoes excretion in its in vivo use in humans and animals because of its poorly absorbing nature. erefore, the metal interaction chemistry of oxytetracycline can be considered an important part of research to standardize its physical and biological profiles.
For the continuation of our ongoing antibiotic research, this work focused on the synthesis of heteroleptic complexes using oxytetracycline and salicylaldehyde along with metal salts. e complexation behavior was investigated by physicochemical studies such as melting point, conductivity, pH, surface tension, viscosity, and density measurements. eir structural characterization was further investigated by spectroscopic studies such as electronic absorption, FT-IR, NMR, and ESI-MS. e surface morphology by SEM study and thermal stability by TGA/DTA study were performed to parameterize the complexes. e complexes were screened for their in vitro antibacterial susceptibility tests with three clinical pathogens to show their biological significance.

2.2.
Instrumentation. e C, H, and N contents of the complexes were recorded using a Euro-E 3000 microanalyzer. Conductivity measurements were done at 25°C in DMSO solvent using an auto-ranging/TDS meter TCM 15+ digital conductivity meter. e VEEGO ASD-10013 programable apparatus was used to record the melting point of the complex. At 25°C ± 0.1°C, the pH measurement was calculated using a digital pH meter AN ISO 9001: 2008 certified company instrument. Using the ring detachment technique, surface tension was measured using the help of Kruss K20S Easy Dyne Force Tensiometer. A PerkinElmer Spectrum II instrument was used to record the FT-IR spectra using KBr pellets in the wavenumber between 400 and 4000 cm −1 . At room temperature, NMR spectra were recorded from Bruker AvII-400 MHz spectrometer using the solvent DMSO-d 6 and TMS (tetramethylsilane) as the reference standard. UV/Vis spectral bands at 10 −3 M concentration in DMSO were calculated using an instrument called Varian Cary 5000 in the range of 200 to 1000 nm. ESI-MS spectrometry technique is applied to record the mass spectra by using a water UPLC-TQD mass spectrometer. A PerkinElmer Diamond TG/DTA instrument was used to evaluate the thermal and kinetic properties of the complexes at room temperature to 1000°C under a nitrogen atmosphere with a linear heating rate of 10°C/min. A JEOL JSM-6390LV scanning electron microscope instrument was used to detect the surface morphology. e geometry optimization of the complexes was done with the help of 3D modeling via Chem3D Pro.12.0 software.

Synthesis of Complexes.
e metal complexes Cd-OTC/ Sal and Mo-OTC/Sal were prepared by heating the mixture solution of 20 ml oxytetracycline hydrochloride (0.9941 g, 2 mmol) in ethanol with 10 ml aqueous solution of CdCl 2 .H 2 O (0.4029 g, 2 mmol) /10 ml alcoholic solution of MoCl 5 (0.5469 g, 2 mmol). To this solution, 0.2 ml of salicylaldehyde (2 mmol) was added and refluxed for 8 h. Ammonia solution was added dropwise to maintain a pH of 7. Under these conditions, precipitation of the complexes was formed and filtered, then washed with ethanol, and finally dried under vacuum desiccators over anhydrous CaCl 2 . e precipitate was then kept in an airtight vial for further use. e synthetic route for the metal complexes is shown in Scheme 1.

Antibacterial Assessment.
e synthesized metal complexes were tested for their in vitro antimicrobial assessment which was done at the Microbiology Laboratory of MMAM Campus, Tribhuvan University, Biratnagar. e tests were performed by modified Kirby-Bauer paper disc diffusion on three pathogens: S.aureus (Gram-positive) and E. coli and P mirabilis (Gram-negative). e culture of bacteria was revived by inoculating the organism in freshly prepared nutrient agar and kept in an incubator at 37°C for a few hours for complete growth. For the tests, test solutions were prepared by dissolving the synthesized complexes in 30% DMSO at three different concentrations (50, 25, and 12.5 μg/μL) and blank paper discs of 5 mm diameter size with Whatman No. 1 filter paper cut by a punching machine and sterilized in an autoclave. e MHA media was prepared in an autoclave and solidified on Petri discs under UV laminar flow to decrease bacterial contamination. e fresh revived bacterial culture was spread on the solidified MHA media, and blank sterilized discs were also seeded and loaded with 10 μL test compounds under UV laminar flow to decrease their bacterial contamination. One blank disc soaked with DMSO acted as a solvent control while another amikacin (30 μg/disc) acted as a positive control to compare the effectiveness of the tested compounds. After performing all these tasks, the loaded Petri plates were placed in an incubator for up to 24 h at 37°C to note the diameter of the zone of inhibition measured by the help of the antibiogram zone measuring scale [19,20]. and air-stable and have greater melting points. All the complexes were soluble in DMSO and DMF, but insoluble in water. e complexes were stored in an airtight vial and kept in vacuum desiccators under anhydrous CaCl 2 . e change in the color of the ligand up to complex formation is the result of the complexation of the ligand with metal ions, which is further supported by pH, conductivity, surface tension, density, viscosity, and melting point. e TGA/ DTA and electronic absorption denote better results of the calculated value and conclude a better relationship with the proposed structure.

FT-IR Spectral Study.
Characteristic IR bands for the metal complexes and their assignments are presented in Figures 2 and S1. e FT-IR spectra of the Cd-OTC/Sal and Mo-OTC/Sal metal complexes showed characteristic bands at 3432 cm −1 and 3437 cm −1 , which may be assigned to the υ(OH/NH) stretching vibration [21,22]. Similarly, the -CH 3 stretching vibrations appeared at 2924 cm −1 and 2928 cm −1 region [23,24]. e strong intensity bands at 1599 cm −1 and 1628 cm −1 are due to the carbonyl (C�O) group band, which partially overlaps the N-H band appearing as a small doublet [25,26]. A band corresponding to the υ(C-N) stretch appeared at 1452 cm −1 and 1456 cm −1 and was displaced by coordination with the metallic center. Such behavior of a band was also found in the literature [27]. At a lower frequency level, the metal complexes show bands at 595 cm −1 & 517 cm −1 and 503 cm −1 & 463 cm −1 , which may be due to the υ(M-O) and υ(M-N) coordination modes, respectively [28,29].

1 H-NMR Spectral Study.
For further confirmation of the binding mode of metal ions in the complexes, the 1 H-NMR spectrum in DMSO-d 6 solvent was carried out at room temperature using TMS (tetramethylsilane) as a reference standard. e chemical shifts (δ) are measured in ppm units present in the downfield from TMS [30,31]. e 1 H-NMR spectra of the metal complexes are presented in Figures S2  and S3, and the spectral data are presented in Table S1. e Cd-OTC/Sal showed spectral peaks at 6.889-7.355 ppm, attributed to aromatic protons. ese aromatic protons are slightly moved up-field and support their coordination of ligands with metal ions [32]. e peaks in the region of 4.284-4.397 ppm suggest the peak for the -NH group [33]. A singlet peak at 1.670 ppm in the spectra of the metal complex was assigned to the methyl protons. Similarly, the peaks at 2.370-2.553 ppm are assigned to methylene protons [31].
e Mo-OTC/Sal complex shows a multiplet peak at 7.186-7.205 ppm, assignable to aromatic protons [34]. e peak at 1.047-1.082 ppm is due to a methyl group and a singlet peak at 2.551 ppm is assigned to methylene protons [35]. Each hydrogen of the aromatic ring observed one distinct peak at 4.414 ppm [36]. Hence, the values in the peaks show good agreement with the proposed structure of the metal complexes.

Mass Spectral Study.
e ESI mass spectrometry is an instrumental technique for determining the molecular mass of compounds. It helps to determine the composition and purity of the compounds. e mass spectrum provides information about the stoichiometric compositions of the compounds. In our study, the mass spectral peaks at m/z 693 and 747.41 amu for the respective Cd-OTC/Sal and Mo-OTC/Sal complexes are assigned to the molecular ion peak [M+H] .+ , and their corresponding base peaks are at m/ z � 461 and m/z � 616, which signify the proposed molecular formula of the complex [37,38]. Besides the molecular ion peak, there are additional peaks called fragment peaks formed by fragmentation of molecular ion peaks and lie in the region at m/z 690, 620, 543, 483, 385, and 300 in the Cd-OTC/Sal complex, and at m/z 743, 728, 723, 709, 674, 640, 537, 473, and 450, respectively, in the Mo-OTC/Sal complex. e ESI-MS spectra are presented in Figures 3 and S4.

Electronic Absorption Spectral Study.
e important electronic absorption spectral bands for metal complexes were recorded in the 250-800 nm ranges of wavelength at room temperature in DMSO solvents taking the same solvents as the blank [30]. Electronic absorption spectroscopy is an analytical instrumental tool for determining the characterization and identification of binding modes for compounds. e metal complex of Cd-OTC/Sal shows peaks at 267, 322, and 375 may be due to π ⟶ π * , n ⟶ π * transitions. e electronic configuration of the Cd(II) metal complex was d 10 , which signifies the absence of any d-d electronic transition and the bands attributed to the CT (charge transition), compatible with tetrahedral geometry; hence, their absorption band spectra contain red and blue shifts with hyperchromic effect ions [21,38]. In the same way, the metal complexes of Mo-OTC/Sal showed peaks at 264 and 318 nm, signifying the π ⟶ π * and n ⟶ π * transitions due to the -C�N-and -C�O-groups in the ligand. Band ∼318 is denoted as the characteristic band of the ligand. Since the complex is diamagnetic and has an octahedral geometry, it is assigned as a ligand-metal charge transfer existing from the HOMO of phenolic oxygen to the LUMO of the molybdenum [39,40]. All these bands are presented and assigned in Figure S5.

ermal Study.
TGA/DTA studies are used to determine the composition of materials and also to predict the thermal and kinetic stability of compounds. is analysis was done in the temperature range of 40°C to 860°C under a nitrogen atmosphere at a linear heating rate of 10°C/min. TGA/DTA is also an analytical instrumental technique that determines weight loss or gains due to various processes such as absorption, adsorption, sorption, or desorption of volatile components, decomposition, oxidation, and reduction reactions. e TGA curves help the scientist for profile structural information for new compounds having a coordination sphere. e initial decomposition steps in complexes are an endothermic process that denotes the loss of water molecules [41,42].

Kinetic Parameters.
e thermodynamic and kinetic parameters of the complexes were obtained using the following Coats-Redfern relation: where T represents the temperature observed on the DTG curve, A and E * denote the Arrhenius pre-exponential factor and activation energy, which can be calculated from the graphical process. R denotes the universal gas constant and β is the linear heating rate. Using the equation y � mx + c, a linear plot on the left-hand side vs. 1/T, whose slope (−E * /R) gives the activation energy. Similarly, other kinetic parameters such as the entropy of activation (ΔS * ), enthalpy of activation (ΔH * ), and free energy of activation (ΔG * ) were calculated using the following equations: Here, various decomposition steps of the kinetic and thermodynamic parameters were calculated and are presented in Tables 1 and 2 [43]. All the complexes show greater values and reflect their high thermal stability because of their covalent bond character. In both complexes, all decomposition steps contain negative entropy of activation, which clearly shows a nonspontaneous dehydration reaction, and the positive values of ΔG * of all decomposition stages for both complexes show nonspontaneous nature and ΔH * is negative which indicates the exothermic process, and the correlation coefficient is shown in the graph indicating a better fit.

SEM Study.
e coordination of metal with the ligand changes the metal complex surface morphology and was done by SEM (scanning electron microscopy) analysis. SEM is an important analytical instrumental technique for surface morphology. e micrograph shows the size, shape, ductility, strength, and arrangement of an object. e SEM micrograph of Cd-OTC/Sal metal complex (Figure 5(a)) exhibits microsphere-like morphology with aggregation of small nanoparticles, leading to the formation of a large sphere. Similarly, Mo-OTC/Sal metal complex ( Figure 5(b)) shows that the surface morphology is a spherical nanomaterial whose aggregation gives rise to an increase in the size of a large sphere [44][45][46], as presented in Figure 5.

Molecular Modeling Study.
e synthesized complexes were geometrically analyzed and characterized by molecular simulation processed in CS ChemOffice 3D Pro.12.0 version software, which gives a better and more accurate assessment of the theoretical predictions and proposed structure of molecules. By using the MM2 program, the optimized structure of the complexes was predicted. Energy optimization was done repeatedly to obtain the minimum energy for the proposed geometry [47,48]. e Cd-OTC/Sal and Mo-OTC/Sal metal complexes were reported to have tetrahedral and octahedral geometries with final geometrical energies of 923.1740 and 899.3184 kcal/mol, respectively. By performing the calculation, bonding parameters such as bond length and bond angle with their optimized 3D molecular structures were obtained and are presented in Table S2 and Figures 6 and 7. rough this discussion, the structure of the metal complexes was obtained. e potential energy is the sum of all different types of energy:   Journal of Chemistry subscripts signify the bond stretching, angle bending, deformation angle, van der Waals interactions, out-of-plane bending, simple bending, and electronic interaction, respectively.
3.6. Antibacterial Sensitivity Study. Metal complexes (Cd-OTC/Sal and Mo-OTC/Sal) were screened for their antimicrobial evaluation using the modified Kirby-Bauer paper       Table 3, and the pictorial representations are reported in Figures 8-10 and S7. e study revealed considerable antibacterial potency of the complexes with all bacterial pathogens. However, the parent drug oxytetracycline has shown a greater inhibitory effect relative to complexes. Moreover, the growth inhibitory effect is greater at higher concentrations of the complexes [49,50]. Antibacterial potency of the complexes is based on the chelation theory. Chelation provides stability of the complex and also provides access for easy permeation of complex through the lipid layer of organisms. is helps in rupturing of the cell wall and deactivates bacterial action.

Conclusions
In summary, the Cd-OTC/Sal and Mo-OTC/Sal complexes were prepared successfully by coordination of metal ions with oxytetracycline and salicylaldehyde ligands. ey were characterized using various spectral and physicochemical techniques. e spectral analysis revealed coordination of metal ions through O at C3 and the amide N atom of C2 of ring A of oxytetracycline and O atoms of salicylaldehydes. e complexes were found as amorphous and colored solids that are insoluble in water but soluble in DMSO and DMF. e electronic absorption data concluded the tetrahedral and octahedral geometries of the Cd-OTC/Sal and Mo-OTC/Sal complexes.
is was further supported by molecular modeling studies. Antibacterial studies revealed significant antibiotic action against S.aureus, E.coli, and P. mirabilis bacterial pathogens. e study showed a better antibiotic effect of Cd-OTC/Sal compared to Mo-OTC/Sal.

Data Availability
e authors share the data underlying the findings of the manuscript.     Figure S1: FT-IR spectra of Mo-OTC/Sal metal complex. Figure S2: 1 H-NMR spectra of Cd-OTC/Sal metal complex. Figure S3: 1 H-NMR spectrum of Mo-OTC/Sal complex. Figure S4: ESI-MS spectrum of Cd-OTC/Sal complex. Figure  S5: electronic absorption spectrum of complexes. Figure S6: thermogram of Cd-OTC/Sal complex. Figure S7: antibacterial activity showing growth inhibition zone around the loaded disc.