Antibacterial Activity of 2-Picolyl-polypyridyl-Based Ruthenium (II/III) Complexes on Non-Drug-Resistant and Drug-Resistant Bacteria

A new hexadentate 2-picolyl-polypyridyl-based ligand (4, 4'-(butane-1, 4-diylbis(oxy))bis(N, N-bis(pyridin-2-ylmethyl)aniline)) (2BUT) (1) and its corresponding Ru(II/III) complexes were synthesized and characterized, followed by assessment of their possible bioactive properties towards drug-resistant and non-drug-resistant bacteria. Spectroscopic characterization of the ligand was done using proton NMR, FTIR, and ESI-MS, which showed that the ligand was successfully synthesized. The Ru(II/III) complexes were characterized by FTIR, UV/Vis, elemental analysis, proton NMR, ESI-MS, and magnetic susceptibility studies. The analysis of ESI-MS data of the complexes showed that they were successfully synthesized. Empirical formulae derived from elemental analysis of the complexes also indicated successful synthesis and relative purity of the complexes. The important functional groups of the ligands could be observed after complexation using FTIR. Magnetic susceptibility data and electronic spectra indicated that both complexes adopt a low spin configuration. The disc diffusion assay was used to test the compounds for antibiotic activity on two bacteria species and their drug-resistant counterparts. The compounds displayed antibiotic activity towards the two non-drug-resistant bacteria. As for the drug-resistant organisms, only [Ru2(2BUT)(DMF)2(DPA)2](BH4)43 and 2, 2-dipyridylamine inhibited the growth of MRSA. Gel electrophoresis DNA cleavage studies showed that the ligands had no DNA cleaving properties while all the complexes denatured the bacterial DNA. Therefore, the complexes may have DNA nuclease activity towards the bacterial genomic material.


Introduction
Ever since the success of cisplatin as an antitumor agent, other platinum group metal complexes have gained a lot of interest as potential bioactive agents, especially those of ruthenium [1][2][3][4][5][6]. Over the years, ruthenium complexes were observed to have bioactive properties as potential antimicrobial, anticancer, and antiviral agents [7][8][9][10]. Ruthenium does not have any specific biochemical role but is still not toxic in biological systems [1]. Ruthenium complexes have also gained much interest because of their special chemical properties such as having similar ligand exchange properties to Pt (II) complexes [2]. erefore, some complexes of ruthenium would have similar bioefficacy to platinum complexes, although having lower cytotoxicity as opposed to platinum complexes [2]. Furthermore, ruthenium can access variable oxidation states (II, III, and IV). e interconversion potential associated with these ruthenium oxidation states is known to be relatively low. us, changing through these oxidation states in biological systems may not be difficult. Also, the low toxicity of ruthenium complexes has been observed to come from the inert Ru(III) complexes which have been shown to become active when reduced to Ru(II) by oxidative reductive biochemical environments such as cancer cell environments [11][12][13].
Various literature reports have demonstrated that ruthenium complexes bearing pyridyl moieties have potent antibacterial activities and this has been associated with variable oxidation states, the low toxicity of the ruthenium centre, and the ability of pyridinyl rings to penetrate membrane walls of bacteria. A few examples such as those with 1, 10-phenanthroline and guanine ligands are known to exhibit some antibacterial activity towards Gram-positive and Gram-negative bacterial strains. ese complexes showed biological activity towards Gram-positive and Gram-negative bacterial strains. eir antibacterial activity against the multidrug-resistant (MDR) Klebsiella pneumoniae is reported to be even higher than that of commercially available antibiotics chloramphenicol and ciprofloxacin [3]. In another study by Gopinath et al., green chemistry synthesis of ruthenium nanoparticles (NPs) using leaf extracts of Gloriosa superba plant was carried out. ese rutheniumbased green NPs exhibited some interesting broad-spectrum antibacterial activity towards both Gram-positive and Gram-negative bacteria [4]. Lam et al. developed some Ru(II) bis(2, 2′-bipyridyl) complexes with N-phenylsubstituted diazafluorenes and tested their bioactivity towards methicillin-resistant Staphylococcus aureus (MRSA) [5]. e lower minimum inhibitory concentration of their cis-dichlorobis(2, 2′-bipyridine)ruthenium compared to other complexes against the MRSA suggested that it had higher potency.
Our previous study [6] demonstrated that the polypyridyl ruthenium complexes exhibit antibacterial activity against two non-drug-resistant bacteria S. aureus and Klebsiella pneumoniae. e complexes showed broadspectrum activity by inhibiting the growth of both Gramnegative and Gram-positive bacteria. In addition, all the ligands showed some activity towards the drug-resistant bacteria MRSA. However, no complexes had activity towards both MRSA and MDR Klebsiella pneumoniae. In that regard, we modified the ligands on the ruthenium centre with the hope of targeting the drug-resistant bacteria, and our findings are reported herein, where a dinuclear Ru(II) complex and a ligand displayed interesting antibacterial activity towards drug-resistant bacteria.

Materials and Instrumentation.
Chemicals and reagents used for synthesis and those for biological activity assays were purchased from Merck, Sigma-Aldrich, USA, and used without further purification. e neat film infrared spectroscopy of the compounds was recorded in the 4000-500 nm region using a PerkinElmer System 2000 FTIR spectrometer (PerkinElmer, USA). UV-Visible absorption spectra were recorded using a Shimadzu UV-31-1 PC scanning spectrophotometer (Shimadzu Corp, Japan) in 1 cm path length quartz cells at room temperature. HRESI-MS data were acquired using Bruker Daltonics micrOTOF-Q II ESI-Qq-TOF mass spectrometer (Bruker Daltonics, Germany). e acquisition parameters were as follows: HRESI-MS; drying gas was 50 L/h at 180°C drying gas temperature, the desolvation gas was 591 L/h at 300°C, and capillary voltages are 4.5 kV. 1 H-NMR and spectra were acquired in 5 mm NMR tubes at 298 or 310 K by an Agilent 600 MHz spectrometer in the range 0-10 ppm. Solvents used were deuterated chloroform with the reference material TMS (Agilent Technologies, USA). e data were processed on MestReNova version 9.0.  4 (DPA)] 2 was prepared according to the procedure in our previous study [6]. Briefly, the synthetic scheme for the synthesis of the ligand and all the complexes is shown in Figure 1.

Antimicrobial Studies.
Disc diffusion assay and minimum inhibition concentration methods were followed as in our previous publication [6] and the National Committee for Clinical Laboratory Standards (NCCLS) as summarized in the literature [7]. All the sample organisms were clinical isolates that were kindly donated by the National Health Laboratory, Botswana, Gaborone. e minimal inhibitory concentration (MIC) was determined by the broth microdilution method. Six concentrations of each of the metal compounds were made in serial dilutions: (40, 20, 10, 5, 2.5, and 1.25) g/mL. Bacterial inocula, with an incubation time not over 24 h, were adjusted to the 0.5 McFarland standard and further diluted down to 5 × 10 5 CFU/mL by double distilled water. For the determination of the MIC, serial dilution was made in MH broth to a final volume of 100 μl in 96-well plates, and an aliquot of 100 μl of the bacterial solution was added to each solution. e experiment was done using alamarBlue; this experiment shows a color change. In the color change, the MIC was observed as the first dilution without a color change from the blue solution.
is was observed as the concentration of the metal compounds increases. e color change of the dye turns from blue to pink to indicate live microorganisms. e bioassays were performed in triplicate for accuracy. e bioassays were statistically evaluated using an ANOVA followed by T-test (p < 0.05). e disc diffusion assay was briefly done as follows. Bacterial culture suspension was inoculated on Mueller-Hinton (MH) agar in 90 mm Petri dishes. e bacterial strains used were S. aureus, K. pneumoniae, MRSA, and MDR K. pneumoniae, and quality control bacteria such as Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Klebsiella pneumonia (ATCC 70063) were included in the experimental setup. en, sterile Whatman filter paper discs, impregnated with test compounds (5 μl at concentrations determined from MIC), were placed on the agar and then incubated at 35 ± 2°C for 18 h. For a negative control, a Petri dish containing only the MH culture medium was included. After the 18 h incubation, zones of inhibition including the diameter of the discs were measured. Inhibition zones above 7 mm in diameter were considered as positive results.

DNA Cleavage Assay.
e method used was adopted as described before [6]. DNA binding ability of the test compounds towards the S. aureus bacterial DNA was determined by agarose gel electrophoresis to assess whether the compounds had any interaction with bacterial DNA. Ten microlitres of compounds that exhibited antimicrobial activities as determined in the MIC assay were mixed with 10 μl of 53 μg/mL of the S. aureus DNA in Tris-HCl/NaCl buffer solution and then incubated at 37°C for 2 h. After incubation, the samples were run on 0.8% arose gel in Tris-acetic acid-EDTA buffer, at 60 V for 90 min.
e gel was then stained with ethidium bromide and photographed under 254 nm UV light.

Synthesis of Compounds.
e three new compounds, the ligand 2BUT 1 and complexes 3 and 4 shown in Figure 1, were prepared successfully in moderate amounts. Compound 1 was achieved by chemoselective alkylation of the oxygen nucleophile using 1, 4-bromobutane with 2 molar equivalent of 4-aminophenol to afford 4, 4'-(butane-1, 4diylbis(oxy))dianiline intermediate ligand. is was done to block the competitive reaction between the oxygen and nitrogen nucleophiles of 4-aminophenol. e reaction path for the synthesis of the 2BUT ligand was proposed to follow the S N 2 mechanism. is was suggested because of the use of a strong base sodium hydroxide and the use of a primary benzylic/pyridyl halide 2-picolyl. e ligand was recovered as a brown oil that formed at a yield of 76%. e intermediate ligand 4, 4'-(butane-1, 4-diylbis (oxy))dianiline has four protons that were deprotonated by the strong base sodium hydroxide in order to attach the 2-picolinic arms.
is new attachment of the 2-picolinic arms was observed spectroscopically, where in proton NMR there was an introduction of the aromatic multiplets coming from the 2picolinic arms, while in FTIR there was disappearance of the (NH 2 ) peaks in vibrational frequencies above 3000 cm −1 .
ere was also a new peak of the aromatic nitrogen coming with the 2-picolinic arms observed at 1431.3 cm −1 . e reaction path for ruthenium complexes was proposed to follow the dissociative or the associative mechanism. is was because there was substitution of the ligands during the reaction. Also, the nitrogen-based ligand that was reacting with RuCl 3 •3H 2 O is a borderline base, and Ru(III) is also borderline. is made it possible for either the associative or dissociative mechanism to be favoured. So, in that regard, the pyridyl ligands were bound to the ruthenium centre to form the Ru(III) [8]. As for the Ru(II) complex, by using the reducing agent dimethylformamide as the solvent and excess pyridyl ligands, the ruthenium was reduced to the softer Ru(II) [8]. After many failed attempts to grow crystals, we resorted to using elemental analysis and other complementary data to characterize complexes 3 and 4 and their characterization is reported in the next section.

Characterisation by Fourier Transform Infrared
Spectroscopy. Characteristic vibrational frequencies of the available functional groups were assigned with FTIR. is was done to observe the stretching frequencies associated with important functional groups within the ligands and complexes. e important functional groups to look at in the new ligand as well as the complexes are shown in Table 1.
e intermediate ligand 4, 4'-(butane-1, 4-diylbis (oxy)) dianiline showed the amine (-NH 2 ) vibration at the frequency of 3311-3387 cm −1 , which disappeared upon attachment of the 2-picolyl moieties. e 2BUT ligand showed the following important functionalities that were still observed from the intermediate ligand: the aliphatic (C-H) vibration at a frequency of 2926 cm −1 . e aromatic (C�C) vibrations at a frequency of 1508 cm −1 and the (C-O-C) vibration which is a sharp and strong peak at 1230 cm −1 were also observed. Lastly, we observed the appearance of the aromatic pyridyl nitrogen vibration (C�N) at 1431 cm −1 . e commercially bought 2, 2-dipyridyl amine ligand fundamental peaks are also tabulated in Table 1, to compare with the new 2BUT ligand. e complexes FTIR data indicated that the complexes were formed. is was based on the binding nature of ruthenium to the ligands. According to Hooke's law, vibrational frequency is directly proportional to the strength of the spring.
is means that the stronger the spring, the higher the vibrational frequency. Electron density may increase or decrease on the bonds due to complexation; thus, the vibrational frequency of the bonds may increase or decrease. ere was an observed decrease in the vibrational frequency of the carbon-nitrogen bond of the pyridyl nitrogen, and this was assumed to be caused by the ruthenium inductively pulling electrons from the pyridyl nitrogen as depicted in Figure 2 [6]. All the other important functional groups coming from the ligands in the complexes were observed and tabulated in Table 1.

Electronic Spectra and Magnetic Susceptibility.
e electronic spectra and magnetic susceptibility studies were done to find the oxidation state and the spin of the complexes. Electronic transitions observed below 300 nm were assigned to ligand π-π * and other charge transfer transitions, especially that the pyridyl ligands have low-lying π-orbitals and the metallic ion has high energy unoccupied d-orbitals. However, all transitions observed above 400 nm were assigned to the d-d transitions. e Ru(III) complex 4 was expected to show at least four energetically observable d-d transitions according to its d 5 Tanabe-Sugano diagram. e complex was assigned to be a low spin complex because of the ruthenium 4d orbitals being large; this means that the pairing energy is most of the times smaller than the splitting parameter, thus resulting in low spin complexes with any ligand type. So, using a d 5 Tanabe-Sugano diagram, complex 4 was assigned the d-d transitions 604 nm 2 T 2g (I) ⟶ 2 T 2g , 2 A 2g (I) and 412 nm 2 T 2g (I) ⟶ 2 E g (I) which were the energetically visible bands. ese are summarized in Table 2, with molar extinction coefficients which also suggested that these were d-d transition bands. Furthermore, complex 4 had an experimental magnetic moment of 1.68 BM contrary to a spin-only calculated magnetic moment of 1.73 BM for a low spin d 5 complex. From a low spin d 5 Tanabe-Sugano diagram, it is observed that this complex has a ground term of T, meaning that it should have a spin-orbital contribution as this is true for complexes with T terms. erefore, an effective magnetic moment of above 1.73 BM (calculated spin-only magnetic moment) was expected. However, this deviation can be explained by evoking the Jahn-Teller distortions as depicted in Figure 3.
e ligand used on this complex 4 had a pyridyl moiety which has many bonding interactions that made it above average in strength in the spectrochemical series.
e dorbitals of ruthenium interact with the following: the pyridyl sp 2 hybrid lone pair of electrons in the aromatic nitrogen, the π electrons coming from aromatic conjugated π bonds, and the empty ring π-orbitals may also accept electrons from the metal orbitals too. ese many orbital interactions were assumed to lead to some weak elongated Jahn-Teller distortions [9] on complex 4 d-orbitals, which caused the effective magnetic moment to be lower than the spin-orbit and spin-only calculated magnetic moments. Figure 3 shows how these weak Jahn-Teller distortions may have contributed to this. e proposed structure of complex 4 is assumed to transform under the C s point group. is point group's character table contained the totally symmetric A′ and the antisymmetric A″ symmetry elements.
ese symmetry elements showed that the e g orbitals were totally symmetric and transformed under the A′ symmetry element, while the t 2g orbitals of this complex did not have the same symmetry and transformed under both A′ and A″. Two t 2g orbitals d yz and d xz were observed to transform under the A″ symmetry element. e A′ symmetry element contained the other d xy orbital. From the character table, it showed that the t 2g orbitals that transform under the A″ were not degenerate but were both symmetrically similar.
us, this suggests that these three t 2g orbitals are not on the same energy level, hence suggesting an elongated weak Jahn-Teller effect along the z-axis shown in Figure 3 [9]. is also meant that the electron on the d xy orbital was unable to interact with the other electrons on the other t 2g orbitals to enforce spin-orbit contribution towards the effective magnetic moment of complex 4 [10].
e Ru(II) complex 3 was assumed to be a d 6 low spin complex, and this was observed with an effective magnetic moment of 0 BM. However, according to a d 6 Tanabe-Sugano diagram, at least 2 energetically allowed d-d transitions should be observed with one observable charge transfer band at 345 nm. And according to our observation, these transitions for complex 3 as summarized in Table 2 were at 532 nm 1 A 1g (I) ⟶ 1 T 1g (I) and 462 nm 1 A 1g (I) ⟶ 1 T 2g (I). e calculated molar extinction coefficients of this complex also assume these transitions to be d-d transitions bands.

Elemental Analysis of the Complexes.
Elemental analysis was used to find the empirical formula and purity of the synthesized complexes. e elemental analytical data show that the complexes were successfully synthesized. However, both the complexes contain some water of crystallization in their formula. e Ru(II) complex contains at least 0.9 moles of water while Ru(III) contained at least 0.6 moles of water. e values for the experimental and calculated elemental analysis are shown in Table 3.

Mass Spectrometric Characterization.
A soft ionisation mass spectrometric technique, the electrospray ionization was used, and this was chosen due to the bulkiness of the test compounds. is type of technique has been successful in quantifying the masses of compounds with weak interactions because of its low-energy ionisation. From the data, it was observed that the experimental molecular ion of the test compounds was close to the calculated molecular masses. All the compounds were run under positive ion    Note. e diagram is not to scale. e diagram demonstrates that the e g orbitals transform under the a′ symmetry label while the t 2g orbitals transform under the a′, a″, and a″; this observation suggests the weak Jahn-Teller distortion. is explains why the observed effective magnetic moment was a bit lower than the expected magnetic moment which is because of the unpaired electron not having orbital contribution from interacting with the other orbitals, thus having no effect on the magnetic moment.  Table 4 shows these data.

Proton NMR Characterization.
One-dimensional proton NMR was used as a characterization tool to study the chemical shifts on the ligand proton NMR compared to that of the complexes. Considering the spectrum of the ligand 2BUT, the ligand molecule shows that there might be symmetry between the central (-CH 2 ) groups on the central aliphatic diether chain, suggesting that the two sides of the molecule have similar chemical environments. Even the proton NMR spectral data showed only the chemistry of one side of the molecule. e 2BUT ligand has a para-substituted benzene aromatic ring that was expected to show a doublet of a doublet splitting pattern, which was observed at 6.52-6.68 ppm in the spectrum. e chemical shift data are displayed under Section 3.2. e 2BUT ligand showed eight observable protons from the aliphatic chain. Of these eight protons, four of each set showed the same chemical environment, therefore giving similar splitting which resulted in only two peaks. e four protons in the central sp 2 carbons of 2BUT were coupled to the outer four protons within the central aliphatic chain leading to a quintet splitting pattern observed at 1.75-1.82 ppm. e other four outer protons which were on the carbon atoms directly bound to the oxygen had only two proton neighbours each. us, they showed a multiplicity of a triplet at 3.82-3.84 ppm. Furthermore, analysis of the 2BUT ligand 2-picolinic arms showed a total of four sp 3 protons for each arm. ese protons showed a multiplicity of a singlet since they are not split by any neighbouring protons. e splitting patterns of the aromatic pyridine moiety were similar for both rings as they shared a similar chemical environment. In addition, integration data also supported that there was symmetry in 2BUT. is is also evident from the protons in the aliphatic region which integrate to two protons each, contrary to the expected four protons. As for the central aromatic ring and the region from the (−CH 2 ) in the picolinic arm to the pyridyl ring, integration data also showed symmetry as these protons only showed as one symmetric pair. An example is the central aromatic ring protons which integrate to one while there are two of each of their kind in the whole molecule.
After complexation, proton NMR was further applied in the characterization of the complexes. However, due to the poor solubility of the complexes, the spectral data were not good enough to be used to fully characterize the complexes. e complexes spectra were dominated by the solvent peaks which suppressed the analyte peaks. erefore, other characterization techniques were used to conclusively characterize the complexes. e chemical shift data are displayed under Section 3.2 of this paper. In addition to the poor solubility of the complexes, the Ru(III) complex [Ru 2 (2BUT)(Cl) 6 ] was also paramagnetic.
is paramagnetism resulted in poor resolution of the multiplicity of the chemical shift of the Ru(III) complex and this was more observable in the aromatic region than in the aliphatic region. Using the aliphatic region, it was observed that the ligand had lost its planarity upon complexation with ruthenium. e chemical shift of the 2-picolinic arms of the free ligand was observed at 4.67 ppm, and upon complexation to Ru(III), they shifted to 2.60 ppm which indicated a loss of planarity. As for the Ru(II) complex [Ru 2 (2BUT)(DMF) 2 (DPA)](BH 4 ) 4 , it also showed poor multiplicity, which could be attributed to poor solubility. However, some notable chemical shifts could be observed, for example, the dimethylformamide peaks were observed at chemical shifts of 0.81-0.85 ppm. In this complex, the planarity of the ligand was also lost, as is evident from the aliphatic protons which were observed in different chemical shifts as compared to the free ligand. Nonetheless, since the Ru(II) is diamagnetic, the chemistry around the aromatic region was detectable.

MIC Assay.
e synthesized ligand and the corresponding Ru(II/III) complexes were evaluated for their antibiotic properties against four bacterial species: S. aureus, K. pneumoniae, MRSA, and MDR K. pneumoniae. Following the lack of antibacterial activity of the complexes towards drug-resistant bacteria in our previous work [6], the Analytical data indicate the complexes only had impurity of water. e data also showed that the complexes' empirical formula is similar to the calculated data with very low differences. e mass spectrometric data indicate successful synthesis of the compounds. All the compounds were run on a positive ion mode with molecular ion peaks being observed with proton and metal adducts. modified analogue complexes reported herein were investigated for antibacterial activity towards the mentioned bacteria as in the previous work. We observed that the new analogues had broad-range antibacterial activity on both Gram-positive and Gram-negative non-drug-resistant bacteria. is was explained by the MIC data depicted in Table 5 and the disc diffusion assay data shown in Table 6. e MIC assay showed that there was comparatively more inhibitory activity towards the Gram-positive than the Gram-negative bacteria. As in our previous work, we suggest that this could be attributable to the differences in the Gram-negative bacterial cellular membranes compared to the Gram-positive, where the former uses a porin or efflux pumps in the cell membrane for ingestion and excretion. ese porins may affect what enters the bacterial cells according to the size of the foreign object, and in this case, we suspected the compounds were affected by this [6,11,14]. All compounds had activity towards all the non-drug-resistant bacteria. However, the complexes had superior activity than the ligands. is is because the complexes had better activity at low concentrations, with the exception of the ligand 2BUT having exceptional potent activity towards S. aureus. Following the restructuring of the complexes in [6], the antibacterial activity was observed to be significantly different in the new analogues. e complexes in this work were of a dinuclear nature as compared to the mononuclear complexes in [6] as shown in Figure 4. Although dinuclear complexes have not been studied extensively compared to their mononuclear counterparts, they present interesting chemistry that has only been unveiled recently [12]. e superiority of dinuclear metal complexes has previously been explored in the context of potential anticancer and antimicrobial agents [13]. In the present study, it is likely that the dinuclear nature of the complexes favours their interaction with biological molecules to the detriment of bacterial cells.
is is also suspected to be due to the dinuclear complexes having doubled the capabilities of the complexes on the activity. e neutral complex 4 showed activity towards S. aureus and K. pneumoniae, and this was the case for the analogue complex 5 from our previous work. e dinuclear complex 4 showed better activity as compared to complex 5 where the MIC for S. aureus was 7.50 mg/mL compared to 10.00 mg/ mL. However, the MIC for K. pneumoniae was 10.00 mg/mL for both complexes. e charged complex 3 in this work showed better activity compared to complex 6 from our previous paper. However, for S. aureus, similar activity was observed from the analogues. e dinuclear complex displayed better activity for K. pneumoniae as opposed to the mononuclear analogue. Interestingly, complex 3 had some activity towards MRSA as opposed to its analogue complex 6. Also, the ligand DPA showed activity on MRSA. Complex 3 had bactericidal activity towards MRSA, which means that the complex was able to kill off the bacteria, while the DPA ligand had a bacteriostatic activity towards MRSA, implying that the ligand was able to stop the reproduction of the bacteria. Furthermore, the data indicated that the main ligand for complex 3, 2BUT, still showed no activity towards MRSA. As for the observed activity of 2BUTon S. aureus and no activity on MRSA, it was inferred that this compound had a similar mode of action towards these two bacteria. ese modified dinuclear complexes were suspected to possess better activity due to their structural characteristics. e ligands found in these complexes have planar aromatic rings (the pyridyl moieties and the aromatic ether-amine central ring) which may have induced better affinity to physically bind to biological molecules [15]. Furthermore, the dinuclear nature of complexes 3 and 4 may have greatly influenced their antibacterial properties.
is is because the introduction of the less polar aliphatic chain bridge may have increased the lipophilicity of the ligands and ultimately the whole complex [16]. In that case, it would be possible that there was better interaction and easier access into the bacterial cells due to this phenomenon. Unfortunately, there was still no activity from all the compounds on MDR K. pneumoniae.

Disc Diffusion Assay.
After determining the lowest bioactive concentration of the compounds, disc diffusion was used to find the extent to which the bacterial colony growth is affected by the compounds. Just as in the MIC and even in the previous work's analogue complexes [6], there was an observed high activity of the complexes on the Grampositive S. aureus as opposed to the Gram-negative K. pneumoniae. No activity was observed for MDR K. pneumoniae from all the compounds. However, in terms of MRSA, there was still some activity from complex 3 and the DPA ligand, just as seen on the MIC data. Furthermore, it was still interesting that there was no activity evident for complex 3's major ligand 2BUT and even its analogue complex 6 from the previous work. In that case, we suspect that, upon coordinating 2BUT with ruthenium, the 2BUT ligand became enabled or complex 2 which had no 2BUT ligand became enabled when coordinated with the 2BUT ligand, consistent with previous studies [17][18][19][20]. As for the analogue complex 6, we suspected the introduction of the bridge in complex 3 may have induced the bioactivity of this complex towards MRSA. e zones of inhibition for all compounds are given in Table 6. Notably, 2BUT had some activity towards S. aureus, suggesting that the mode of action may be the same as that of MRSA, which may explain the lack of activity from this ligand on MRSA.

DNA Chelation Assay.
is assay was used to assess the DNA cleaving properties of the compounds. All the compounds which had activity towards S. aureus and MRSA were tested to see if they could cleave DNA into different forms.
e DNA used had been freshly extracted from S. aureus. Figure 5 shows the interaction of these compounds with the DNA.
Lane L represents the DNA marker, while C is the untreated control DNA. Even though ligands showed good activity with the other assays, in this assay the ligands did not show any DNA cleavage. e 2BUT and DPA ligand are on lanes 3 and 4. ese findings suggested that the 2BUT and DPA ligands had different modes of action contrary to the complexes. We concluded that the introduction of the metal centre to the ligands may induce more modes of action or disable the ligands, hence the observation on the gel. e complexes on lanes 1, 6, and 8 showed interesting results. Just as observed from our previous research [6], these complexes also had potent activity towards DNA. We noted that the complexes completely denatured the DNA. We had suggested and still suggest that the strong activity was due to the strong affinity of the complexes towards DNA. e labile ligands (chlorido and dimethylformamide), planar heterocyclic polypyridyl ligands, and the ruthenium metal centre  Ru-DMF Figure 4: e mononuclear ruthenium (II/III) complexes reported in our previous work [6]. e difference with these analogues is that the complexes (3 and 4) in this paper are bridged by a four-member aliphatic chain. e chain in the mononuclear complexes does not connect to anything. are assumed to be the contributing factors to the observed DNA denaturing activity [6,[21][22][23]. ese data suggest that DNA nuclease/denaturing may be the mode of action for the complexes, especially that they showed bactericidal concentrations.

Conclusions
It may be concluded that a new ligand 2BUT and 2 new complexes were successfully synthesized and characterized spectroscopically through FTIR, UV/Vis, elemental analysis, and magnetic susceptibility. e complexes UV/Vis data showed the energetically observable transitions associated with the complexes. e data suggested the complexes to adopt a low spin d 5 and d 6 Ru(II/III), respectively. Magnetic susceptibility studies also suggested the same spin for the Ru(II/III) complexes. FTIR showed all the complexes' important functional groups in the near IR.
ere was an observed decrease in some metal to ligand vibrational bands which indicated coordination of the metal to the ligands. Mass spectrometry and elemental analysis showed the compounds were successfully synthesized. Proton NMR showed that the ligand was successfully synthesized. However, the complex proton NMR was challenging to assign but did prove they were formed. e biological assays showed that complexes and ligands were indeed bioactive. Disc diffusion assay showed that there was more activity towards the Gram-positive bacteria. We also observed that there was more activity against the non-drug-resistant strains S. aureus and K. pneumoniae. ere was some notable activity towards MRSA from complex 3 as opposed to the monometallic complex 6 in our previous work. e DPA ligand also showed some activity towards MRSA. However, all the complexes showed no activity towards MDR K. pneumoniae just like in the previous paper. DNA gel electrophoresis showed that the complexes had potent DNA chemical nuclease/denaturing; this was then suggested to be the mode of action of the complexes or one of the modes of action that could be observed from the complexes. Also, it was observed that the modifications of the analogues of these complexes in our previous work did indeed show some new activity for the complexes.
Data Availability e characterization and biological assay data used to support the findings of this study are included within the article, summarized in tables and under materials and methods. However, there is a supplementary material data sheet that includes the spectral data of the compounds from Figure S1 to Figure S13.  Figure S1: proton NMR spectrum of 2BUT 1. Figure S2: FTIR spectrum of 2BUT 1. Figure S3: HRESI-MS spectrum of 2BUT 1. Figure S4: FTIR spectrum of complex 3. Figure  S5: UV/Vis spectrum of complex 3. Figure S6: HRMS spectrum of complex 3. Figure S7: HRMS enlarged spectrum of complex 3 at assigned molecular ion m/z 1372.7879. Figure S8: proton NMR of Ru(II) complex 3. Figure S9: FTIR spectrum of complex 4. Figure S10: UV/Vis spectrum of complex 4. Figure S11: HRMS spectrum of complex 4. Figure S12: HRMS enlarged spectrum of complex 4 at assigned molecular ion m/z 1104.5452. Figure S13: proton NMR spectrum of the Ru(III) complex 4 (). (Supplementary Materials)