2.1. Materials

All the chemicals used for the synthesis of the complex were products of Sigma Aldrich Chemical Co., USA, except Triphenylphosphine that was supplied by Alfa Aesar, UK, and were of analytical reagent grade.

2.2. Physicochemical methods

Infrared spectra were recorded on Bruker FT-IR spectrophotometer using potassium bromide pellets of all the compounds. Electronic spectral analysis was conducted using UV-visible spectrometer (Cary 5000, US). The decomposition temperature was recorded using an electrically operational melting point apparatus (Kumar Industries, Mumbai, India) bearing heating capacity up to 360 °C. NMR, mass spectrometry, and elemental analysis were made available by SAIF-CDRI, Lucknow, India. All solutions were purged with N₂ for half an hour before each experiment.

2.3. Computational methods

The theoretical calculations were run using a licensed Gaussian 09 software package. The complex structure was optimized using LanL2DZ for Ru and 631 g(d,p) for all other atoms invoking B3LYP as functional. The cis-CO–PPh₃ and trans-CO–PPh₃ forms of the complex were evaluated computationally under DFT investigation using the same formalism. After confirming the absence of imaginary frequency in the generated structures, the resultant optimal conformations were made as input files for the further spectroscopic or other electron density computations following the same calculation scheme as described in the optimization [25,26,27].

2.4. Synthesis

3.1. Structural evaluation of the Schiff base

The formation of the Schiff base ligand has been discussed in light of various physicochemical means. From the data formulation of the ligand is in full agreement with the proposed structure. The formula mass is comparable with the results obtained from the respective elemental analysis and mass spectrometry. FT-IR spectral results indicate two significant vibrational modes at 1,610 cm⁻¹ and 1,575 cm⁻¹, for ν_CH=N and ν_C=N, respectively [19,20,27]. Other vibrational signatures important in identifying ν_(OH) at 3,429 cm⁻¹ and ν_C−O at 1,368 cm⁻¹ are also clear. ¹HNMR spectral analysis shows distinctive resonating signals for enolic protons at 16 ppm. Aromatic protons are well distinguishable at 6.4–7.6 ppm. A feeble 8.2 ppm signal may be assigned for > CH=N− proton, 3.8 ppm for −OCH₃ and remaining methyl groups around 2.5 ppm. Similarly, ¹³CNMR shows > C=N and −CH=N− resonating frequency at 163. The existence of C−O enolic is confirmed from the appearance of chemical shift of 172 ppm different from > C=O that appears usually at 180 ppm. δ values of the range of 117–132 show aromatic carbons of varied substitution. A 50 ppm signifies the methyl groups of OCH₃ portion of o-vanillin moiety, while 14.7–14.8 ppm is meant for ethylene diamine methyl groups. The pentet signal of 2.5 ppm in ¹HNMR and a septet signal near 78 ppm denote solvent signals of DMSO-d₆ used in the experiment. The Schiff base related FT-IR, ESI-mass and NMR spectra are shown in Figures S2–S4. Figure 1 shows the proposed 2D structure of the compounds in the present investigation along with the structure of the ligand [19,20,27].

3.2. DFT-based justification of cis versus trans forms of the complex

In order to remain confined to a single isomeric form of the complex, DFT-based evaluation of both the cis- and trans-forms of the complex with respect to (CO)(PPH₃) was first of all determined. The respective optimized structures have been given in Figure 2. The total energy continum (E_total), zero point energies (ZPE) and frontier orbital energies (FOE) show that trans-isoform is thermodynamically more stable as compared to the cis-form. E_total, ZPE, and FOE for cis-complex come out to be −2,275.57 au, 387.09 Kcal/mole, and 3.65 eV (HOMO-LUMO gap), respectively, whereas the calculation for the same descriptors of the trans-isomer shows E_total, ZPE, and FOE to be −2,275.55 au, 386.67 Kcal/mole, and 3.69 eV. Therefore, it is logical to state here that the DFT structural investigation shall involve mainly the data obtained for the trans-isoform of the complex.

3.3. Structural verification of the complex

The consistency of analytical data with the proposed structure (Figure 1) of the complex is clearly showing an excellent agreement with the theoretical outcomes. In the FT-IR spectrum (Figure S5) of respective complex, ν_CO: 1,961 cm⁻¹, ν_Ph(P−Ph): 1,437 cm⁻¹, ν_CH=N: 1,609 cm⁻¹, ν_C=N: 1,574 cm⁻¹, ν_C−O: 1,381 cm⁻¹, and ν_C=C: 1,513 cm⁻¹ shows the excellent binding ability of Schiff base ligand under discussion and the presence of PPh₃ and CO as co-ligands. The resemblance between the experimental and computed IR data may be also seen as shown in Figure S5.

UV-Vis spectroscopic details of the ruthenium(II) complex clearly indicate the excellent mode of binding between target ligand and the metallic core. The central metal has been found to be diamagnetic indicating the presence of ruthenium in II oxidation state. The ground state of ruthenium(II) (t⁶_2g configuration) is ¹A_1g. The excited states corresponding to t⁵_2g e¹_g configuration are ³T_1g, ³T_2g, ¹T_1g, and ¹T_2g in the order of increasing energy. This speculates the existence of four bands corresponding to the transitions from ¹A_1g → ³T_1g, ³T_2g, ¹T_1g or ¹T_2g (in the order of increasing energy). The electronic spectrum of the complex was recorded in DMSO using 1 mM solution. The respective electronic spectrum of the complex shows four to five bands in the region 210–550 nm. The bands appearing at 210 nm and 317 nm may be assigned due to π → π^∗ and n → π^∗ transitions, respectively. The solvent shows a slight effect on the band positions exhibiting bands around 430–550 nm, which could be assigned to charge transfer transition of an electron from the metal t_2g level to an unfilled molecular orbital derived from the π^∗ level of the CO [28]. This assignment is similar to assignments made for similar other ruthenium(II) octahedral complexes [29]. The observed multiple absorptions prove the presence of different acceptor levels in the complex.

TD-DFT electronic spectral speculation displaying nonzero oscillatory strengths (f) show that the results are acceptable and possess considerable absorption peaks. From f values 0.001 to 0.0481 it is clear that the involved transitions are spin allowed transitions and are d-p mixing and charge-transfer type [30]. Theoretical spectrum may be used to validate the experimental observation. It is clear from Figure S6 that the applied theory in evaluating TD-DFT electronic excitation parameters are in good agreement with what was observed experimentally. The calculation involves three levels of excitation involving the respective wavelength, the energy of excitation, and the probability of excitation to occur among all the given possible transitions. It may be mentioned here that total energy, E(TD-HF/TD-KS) = −2275.40 a.u., meant for the state of optimization and/or second-order correction was the over-all result for the single point energy calculation. The theoretical UV-Vis data involving the energies of excitation and the excitation coefficients are given in Table 1. The excitation coefficient serves as a speculative tool to find the most probable transitions possible in the complex. The square of the coefficient multiplied by a factor of 200 gives the percentage contribution of an electronic transition. It is HOMO-LUMO transition that has been computed to be most probable. Other transitions also tend towards visible region. From the frontier orbital analysis (Vide infra) the HOMO-LUMO gap is quite low as compared to the coordinated ligands. Moreover, from the theoretical infrared spectral insight (vide supra) it is Ru−CO bond that needs around 6 KJ/mol only for its bond with metal to vibrate. Hence, one may speculate it here that all the spectral data present a predictive vision that the CO ligand will be experienced as fast and foremost releasing group and that too at the expenses of visible form of energy [31,32,33].

Table 1: Theoretical UV-Vis data of the complex.

Mass spectrum of the complex is shown in Figure S7. The expected formula mass of the compound is directly fetchable from the spectrum. Almost the molecular weight of 702 with a distinctive stretch of certain units certifies the isotopic forms of the complex in respect to Ru element. The m/z value of 312 and 313 may be assigned for the presence of Schiff base ligand. All other fragments having existence below 5% were ignored.

From the NMR analysis (Figure S8) of the complex again the certainty of proposed formula of the complex is established. The absence of enolic signals in the respective ¹HNMR spectrum of complex clarifies the coordination of respective Schiff base. Moreover, the recurrence of important resonating frequencies when compared with the respective Schiff base again stems the successful binding. Similarly, in case of ¹³CNMR spectrum, it details the presence of coordinated carbonyl group and also shows the presence of other carbon atoms. For example, 170 ppm is meant for coordinated CO, 161 denotes the resonance of enolic carbon, and the signals at 12 and 48 show the presence of methyl groups [34].

3.4. DFT-based theoretical studies

The various bond lengths, bond angles, and dihedral angles generated from the equilibrium structure of the trans-form of the complex are given in Table 2. The results obtained for the geometry of the complex are in good agreement with the similar type of metallic systems reported elsewhere [35]. An octahedral geometry is proposed for the complex and is eminent from the optimized parameters. After serious examination of the metallic coordination zone it may be enunciated that Ru−CO link bears different electronic property as compared to other connectivites under question. It clarifies the assumption of Ru−CO tendency to behave as the foremost locus to any chemical change. Refer to Figure 2 for the numerical assignments made for the trans-isomer.

3.5. FMO analysis

Frontier orbitals are very important characterizing tools for chemical reaction [36]. It can help in predicting feasibility of a chemical process. Hence, they are called the Frontier orbitals. The HOMO primarily acts as an electron donor and the LUMO hence the acceptor, and the gap between HOMO and LUMO characterizes the molecular chemical stability [37]. The electronic fillings of MO’s in complex verify their diamagnetic behavior. The frontier orbitals that were computed have been shown along with the energy gaps in Figure 3. From the HOMO-LUMO gap of compound, the energy dissipation may be proven helpful in terms of charge delocalization along alternative signal and double bonds. The persistence of LUMO towards CO ligand may be sought in designing the locus to stimulate the compound to release CO.

3.6. MESP and antibacterial activity

Molecular electrostatic potential surface as obtainable from theoretical graphics helps a lot in understanding the potentiality towards biological relevance [38,39,40,41]. The distinctive red, blue, and neural zones as shown in Figure 4 explain the prediction of this property. Keeping in view the appearance of these three spots on the surface diagram, it is easy to establish the cell permeability of the compound. This can be further explained based on electrophilic and nucleophilic zones within the complex. From Mulliken charge analysis O-6, O-16, O-17, O-26, C-8, C-9, C-10, C-11, C-18, C-19, C-20, C-23, N-21, and N-22 represent components of nucleophilic zone, whereas the rest of the atoms constitute the electrophilic region. Such a potential varying justification helps to explain the reactive behavior towards a biological membrane. The emphasis given upon the electrostatic interaction of Ru-core with regard to cellular DNA binding and cell wall traversing phenomenon can thus be inferred computationally.

3.7. Microbial growth, qualitative and quantitative analysis

The hospital origin samples were collected from Bombay Hospital Jabalpur and were cultured and identified in the Microbiology Department of our university. Disposed catheters and urine samples were collected from UTI-infected persons from Mumbai Hospital, Jabalpur, India. The microbial culture, identification, and antibacterial activity were experimented at Biological Science Department, R. D. University, Jabalpur, India. The results in the form of zone inhibition were measured in mm. The microorganisms present in urine samples of CAUTI infected patients were cultured in the nutrient broth and were differentiated using the gram staining procedure into gram positive and gram negative organisms. The organisms were transferred to cystine lactose electrolyte deficient (CLED) agar medium for further differentiation of urinary organisms. Urine samples were shaken well in their containers for even distribution of bacteria. A calibrated wire loop with internal diameter 3.26 mm that hold 0.004 mL of urine was inoculate into the above media. The inoculums were spread with the wire loop on the media plate. Plates were incubated aerobically at 37 °C for 24 h. Pure isolated colonies were Gram differentiated and then biochemically identified. The biochemical tests that were performed for each organism including catalase activity, indole production test, citrate utilization test, and urease test were done for identification. Results from the present study showed that 85% bacterial isolates comprise of 50% nonpathogenic bacteria, 37% pathogenic bacteria, and remaining No Bacteria Growth (NBG). The sampling showed that S. aureus, E. coli, and P. aureginosa were the main pathogenic isolates having existence as 49%, 38%, and 13%, respectively. Urinary samples and catheters almost acted as equal sources of pathogenic bacteria.

3.8. Antibacterial activity

After confirmation of identity of E. coli (MTCC 1304), the respective microorganism being the main second causative agent of this infection was selected for the study. The in vitro biological screening effects of the complex at different concentrations by agar well diffusion method were carried out at Bioscience Department, R. D. University, Jabalpur, India. All the experiments were made in triplicates to arrive at the minimum inhibition concentration (MIC) values of the complex [42]. The results in the form of zone inhibition were measured in mm for 20 μM, 40 μM, and 60 μM solutions of the complex, parent compound, Schiff base ligand, and standard drug (tetracycline). In order to obtain MIC value, the control DMSO was separately tested that has shown negligible activity. It was observed that the metal complex shows much more activity in comparison to ligand, standard drug (tetracycline), and the respective parent compound due to the enhanced lipophilicity of the complex (Figure 5). The results have been comparatively presented in Table 3. MIC value shown by the complex is thus 30 μM.

Figure 5: Petri-plates showing inhibition zones against E. coli.

Table 3: Antibacterial data of the complex and the standard drug (concentration vs. inhibition zones).

The screening tests have thus shown that metal carbonyl complex possesses the highest activity index as compared to other molecular species under the present investigation. The remarked activity against gram negative species culminates the relevance of polyoxy millieu with the cell wall structure [43,44]. The respective activity index can be calculated by the following relation:

From A.I. value of > 1, it can be concluded that the complex is more active as compared to the standard drug employed in the study. Presence of carbon monoxide together with the bioactive components of the complex can be used in immunotherapy. Antibody collection is a laborious task. However, such compounds are engineered to behave as antibiotics of interesting choice. Further stretch in their composition like immunogenic peptides can be brought up by chemical transformation methods. Seeking drugs under this heading may bring forth great approach to treat multidrug resistant microbes causing nosocomial and community acquired infections. The increasing incidence of drug resistant pathogens has developed interest towards compounds that becoming useful therapeutic tools. Briefly, it may be stated that there is a lot yet to be done to explore anti-infectious potential of the metallic complexes in this regard by altering the nature of ligand around the coordination zone. Such results are comparable to the gasotransmitter models reported elsewhere [40,41,45,46,47,48].