Binuclear Rhodium(II) Complexes With Selective Antibacterial Activity

Binuclear rhodium(II) complexes [Rh2Cl2(μ-OOCR)2(N-N)2] {R = H, Me; N-N = 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen)} and [Rh2(μ-OOCR)2(N-N)2(H2O)2](RCOO)2 (R = Me, Et;) have been synthesized and their structure and properties have been studied by electronic, IR and 1H NMR spectroscopy. Antibacterial activity of these complexes against Escherichia coli and Staphylococcus aureus has been investigated. The most active antibacterial agents against E. coli were [Rh2Cl2(μ-OOCR)2(N-N)2] and [Rh2(μ-OOCR)2(N-N)2(H2O)2](RCOO)2 {R = H and Me} which were considerably more active than the appropriate nitrogen ligands. The complexes show low activity against S. aureus. The activity of the complexes [Rh2(OOCR)2(N-N)2(H2O)2](OOCR)2 against E. coli decreases in the series: R=H≅CH3>C2H5>C3H7≅C4H9. The reverse order was found in the case of S. aureus.


Experimental
Synthesis of the complexes.
The antibacterial activity was evalueted on reference strains Staphylococcus aureus 209P (Oxford) and Escherichia coli K-12 ROW. Nutrient broth and nutrient agar (Biomed, Warszawa) as growth and plating media were used, respectively. The media were supplemented with the tested compounds at suitable concentrations. The growth of bacteria was determined after 24 hours incubation at 37 oC. Antibacterial activity was determined either as MIC (minimal inhibitory concentration) (in solid medium) or by serial dilution in nutrient broth by the method presented in the previous paper [27]. In the former about one hundred colony forming units per plate were plated; in the latter 10 cm3 of broth was inoculated with either 108 or 105 colony forming units.
In experiments on mutagenicity of the complexes the test recommended by Ames and Maron [30] was carried out using TA 97, TA 98, TA 100 and TA 102 as bio-hismutants of Salmonella typhimurium.

Results and Discussion
The [Rh2(RCOO)2(N-N)2(H20)2][RCOO]2 complexes were prepared by reactions between [Rh2(RCOO)4(H20)2 and appropriate nitrogen ligand in ethanol, 2-propanol and 1,4-dioxane. Other alcohols may be also used. The reactions in primary and secondary alcohols should be performed in the presence of air because rhodium(II) complexes during heating in these solvents are reduced to the deep green Metal-Based Drugs also confirmed by the presence of CIRh CT bands in the electronic spectra of these compounds in the 290 255 nm [4,5,31].
In the electronic spectra (Table 1) of these complexes in visible region two bands are observed. Band occurs at about 555 -570 nm, about 20 30 nm lower compared with that for [Rh2(RCOO)4(H20)2 compounds because this band corresponds to the *(Rhz)(y*(Rh2) transition [1 5, 31] and the *(Rh2) levels in [Rhz(RCOO)z(N-N)z(H20)2] 2+ complexes due to the interaction of the orbitals of the Rh 2 core with n* orbitals of nitrogen ligands have lower energy than that of the n* (Rhz)(5.eg) orbitals in [Rhz(OOCR)4(H20)2 compounds. The intense band II at about 408 420 nm was assigned to the allowed charge-transfer transition J(Rhz)*(N-N) [31]. This band obscures the absorption of low intensity attributed to the *(Rhz)Qy*(Rh-O transition observed at 430 460 nm in [Rh2(OOCR)4(H20)2] complexes. The energy of the band strongly depends on the nature of axial ligands and encreases with increasing of the field strength of these ligands. Band remains relatively constant for axial oxygen donor ligands, but is blue shifted with nitrogen donor ligand.    2,1997 Binuclear rhodium (11) Complexes with Selective Antibacterial Activity The IR spectra of complexes with phenanthrolines confirmed that carboxylato ligands are symmetrically bonded to the both rhodium atoms because differences between vas(COO) and vs(COO) are small ( Table 2).
The complexes 1 7 and the ligands (phen and bpy) were tested in vitro for antibacterial activity against Gram-negative Escherichia coli ROW and Gram-positive Staphylococcus aureus 209 P (Oxford) strains ( Table 4). The highest activity against Gram-negative bacteria exhibited complexes 1 5 and compounds 6 and 7 are a little less active, however, they are considerably more active than the appropriate Metal-Based Drugs nitrogen ligands. They are active at concentrations 5.10 -6 10-5 M. All complexes (1 7) show rather low activity against S. aureus.
For some [Rh2(OOCR)2(N-N)2(H20)2](OOCR)2 and [Rh2C12(RCOO)2(N-N)2] complexes characteristic is high selectivity against Gram-negative or Gram-positive bacteria. In general, Gram-positive microorganisms are more susceptible than gram-negative ones, the minimal inhibitory concentrations are usually ten times lower for the former (10-6 M) than for the latter (10-5 M). The activity of the complexes [Rh2(OOCR)2(N-N)2(H20)2]-(OOCR)2 against E. coli decreases in the series: R H CH3 > C2H5 > C3H7 -= C4H9. The reverse order was found in the case of S. aureus. Thus, [Rh2(OOCR)2(N-N)2-(H20)2](OOCR)2 complexes with the most hydrophobic R group are the most active against S. aureus [27], this presumably correlating with penetration. This was also confirmed for analogous complexes containing PhCOO and PhCH(OH)COO ligands [27]. The data given in the Table 4 indicate that the most hydrophilic [Rh2(OOCR)2(N-N)2(H20)2](OOCR)2 complexes are the most active against E. coli.  In order to get more information about the nature of the inhibitory activity of the investigated compounds, we have examined the dependence of survival of E. coli in nutrient broth containing complexes 1 and 4 at 37 0C on time (Fig. 2) It is known that some antibacterial drugs, like -lactamase are active only against growing bacteria [32]. The fast decrease of c.f.c. (colony forming cells) with time indicates that the complexes show bactericidal activity against E. coli K-12 ROW. The bactericidal activity was also confirmed by analogous tests performed in saline containing complexes 1 and 4 (10-5 M) at 37 0C. The results given in Table 5 indicate that in these experiments the decrease of survival is also observed, although this decrease is not so strong as in the case of broth cultures. The effectivity of many antibiotics and chemoterapeutic agents depends on the size of the population of treated microorganisms. Moreover, the probability of growth of the mutated cells increases with inoculum size. Therefore, the survival of E. coli cultures in broth over the population range 105 to 108 cells in the presence of complexes 1 7 has been determined. The results presented in Table 6 indicate that the bactericidal activity of these compounds depends on the inoculum size. The complexes 1, 3 and 5 7 are active at concentration equal to or greater than 5.10-5 M in broth inoculated with 108 cells, while 2 is effective at 2.5.10-5 M. The lowest activity, at this inoculum size, shows complex 4. The complexes 1 7 inhibited the growth of E. coli in broth at concentration at least for one order lower when the inoculum size was 105 cells.  [27] and they can be considered as the antibacterial agents with possible application. Therefore, it seemed interesting to investigate the mutagenicity some of these compounds. The performed tests of Ames (Table 7) indicate that the number of the his+bio + revertants of Salmonella typhimurium strains on plates containing complexes 1 7, 8 and 10 at concentration equal to MIC is n6t greater than that on control plates.   The lower number of revertants in the presence of complexes are to be expected because at MIC concentration of complexes some of the treated cells are killed. It is difficult to determine the fraction of survivals in these experiments because it depends on inoculum size. Nevertheless, the tested complexes seem to be rather safe from the point of view of mutagenicity.