In vitro antibacterial activity of dinuclear thiolato-bridged ruthenium(II)-arene compounds

ABSTRACT The antibacterial activity of 22 thiolato-bridged dinuclear ruthenium(II)-arene compounds was assessed in vitro against Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. None of the compounds efficiently inhibited the growth of the three E. coli strains tested, and only compound 5 exhibited a medium activity against this bacterium [MIC (minimum inhibitory concentration) of 25 µM]. However, a significant antibacterial activity was observed against S. pneumoniae, with MIC values ranging from 1.3 to 2.6 µM for compounds 1–3, 5, and 6. Similarly, compounds 2, 5–7, and 20–22 had MIC values ranging from 2.5 to 5 µM against S. aureus. The tested diruthenium compounds have a bactericidal effect significantly faster than that of penicillin. Fluorescence microscopy assays performed on S. aureus using the BODIPY-tagged diruthenium complex 15 showed that this type of metal compound enters the bacteria and does not accumulate in the cell wall of gram-positive bacteria. Cellular internalization was further confirmed by inductively coupled plasma mass spectrometry experiments. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appears to significantly influence the antibacterial activity. Thus, if overall a decrease of the bactericidal effect with the increase of compounds’ molecular weight is observed, however, the complexes bearing larger benzo-fused lactam substituents had low MIC values. This first antibacterial activity screening demonstrated that the thiolato-diruthenium compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies. IMPORTANCE The in vitro assessment of diruthenium(II)-arene compounds against Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus showed a significant antibacterial activity of some compounds against S. pneumoniae, with minimum inhibitory concentration (MIC) values ranging from 1.3 to 2.6 µM, and a medium activity against E. coli, with MIC of 25 µM. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appear to significantly influence the antibacterial activity. Fluorescence microscopy showed that these ruthenium compounds enter the bacteria and do not accumulate in the cell wall of gram-positive bacteria. These diruthenium(II)-arene compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies, especially the compounds bearing larger benzo-fused lactam substituents.

authors observed an overall decrease of the bactericidal effect with the increase of molecular weight, and the complexes bearing larger benzolactam had low MICs.
1. Are the strains MDR?The nature and resistance profiles of the strains should be included, as the motivation of the work is to combat MDR. 2. Stability studies were done in DMSO rather than biologically relevant solvents like buffers or growth media.It is true that the compounds are insoluble in aqueous solutions, but one could perhaps test stability using a mixed solvent like DMSO-d6/D2O.3. Vague descriptive words should be avoided and substituted with more precise descriptions.For example, in "...very limited solvolysis of the ester bonds...", what constitutes "limited solvolysis"?What percentage of the ester bonds were hydrolyzed? 4. The quality of Figure 6 is very poor.The fluorescence was not visible in any of the images, and no cells were seen at either the white or green arrows.5.In the cellular uptake experiments, after incubating with bacteria, the Ru compounds were washed with aq.0.85% NaCl twice.Since the Ru compounds are insoluble in aqueous solution, this procedure will not remove physisorbed compounds on the bacteria, thus leaving residual compounds on the bacteria.This may explain why there was no dependence of the uptake on the incubation time.Correct washing procedure should be used to ensure that the ICP-MS results are indeed from internalized compounds.6.The purity of commercial antibiotics should be included.
Reviewer #2 (Comments for the Author): review attached

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Comments on Spectrum00954-23
Overall: This manuscript reports on the preparation of a series of diruthenium organometallic complexes employing a variety of functionalized ligands including BODIPY fluorescent probes and some lactam containing rings.
Overall the synthesis of the organometallics is carried out according to synthetic procedures that are standard in this area and the characterization data appears to be consistent with the proposed structures.
The rationale for this entire work is not fully clear -other than in vitro testing of some organometallics.It is far from clear if these molecules would have any utility as a clinical therapeutic.Glaring in the absence is the inclusion of any discussion of the potential human toxicity of ruthenium complexes and the barrier that might generate to any further utility of these molecules.
A major scientific shortcoming of the work reported in the manuscript is any preliminary testing on mammalian cell lines.At a minimum a test in a cell line such as HEK293 would potentially provide some insight into the utility of these organometallic complexes.The time and expenses of mammalian cell line testing is acknowledged but in the case of these organometallics it would be necessary to ensure that this work is viewed with the appropriate impact.A conclusive demonstration of selectivity for bacterial cells over mammalian would certainly provide some helpful rationale for this project.
At present this work offers little of significant impact and would not generate much interest in the medicinal chemistry community.

Specific comments:
Complex stability: the authors carry out a stability study by leaving samples in DMSO-d6 -however it is not clear if this was left in an NMR tube and how it was stored for the intervening 100 days.This is also not a very relevant stability test and perhaps a test with at least some concentration of water (not D2O) present in the DMSO would be more biologically relevant NMR Characterization: Data: In the supporting information the authors report the 13C-NMR chemical shifts to 2 decimal places whereas one decimal place it more appropriate even at 100 MHz.Unless the authors have made changes to the default Bruker settings for collecting a broad-band decoupled spectrum then these should be indicated.
 We agree with this referee that the phrase wording is not optimal.However, it is very difficult to provide precise percentage values.Previous NMR & Fluorescence spectra on many of the reported compounds indeed show a very limited solvolysis (~2%).As reported in Chembiochem, 21 (2020) 2818-2835and in ChemBioChem, 23, e202200536, 2022, BODIPY and coumarin conjugates always exhibited a small bathochromic shift of the fluorescence maximum in EtOH compared to CHCl3 (Δλ ca. 10 nm), and the intense fluorescence intensity change depending on the presence, or not, of the trithiolato di-ruthenium unit could be used to monitor the stability of the conjugates towards hydrolysis and their behavior in vitro.
We have therefore reworded this sentence: "... and for these compounds, fluorescence measurements proved that the ester bonds remained intact for 48 h, but that a very limited hydrolysis of the ester bonds with the release of coumarin dyes after 168 h could be observed".
4. The quality of Figure 6 is very poor.The fluorescence was not visible in any of the images, and no cells were seen at either the white or green arrows.
 We regret that the quality of figure 6 is poor.However, this is not due to the figure itself, but rather to the eps <-> pdf conversion.In fact, the original figure is of good quality and all the elements required for its interpretation are clearly visible.
5. In the cellular uptake experiments, after incubating with bacteria, the Ru compounds were washed with aq.0.85% NaCl twice.Since the Ru compounds are insoluble in aqueous solution, this procedure will not remove physisorbed compounds on the bacteria, thus leaving residual compounds on the bacteria.This may explain why there was no dependence of the uptake on the incubation time.Correct washing procedure should be used to ensure that the ICP-MS results are indeed from internalized compounds.
 The water solubility of the Ru-compounds investigated, while generally low, varies widely from one compound to another.Indeed, the solubility of the unsubstituted Ru-compounds 5 & 6 can reach 0.5 mM (as reported in one of our first studies, ref 38, Inorg. Chem, 2011, 50, 10552), while the solubility of compounds 9-10 bearing an alkyl chain or conjugates 13-16 having a BODIPY group is effectively almost zero.As a result, we do not believe that hypothetical physisorbed compounds may explain why we did not observe a dependence between the uptake and the incubation time.For instance, the ICPMS provided the same value for the moderately water-soluble compound 5 and the insoluble BODIPY compound 15.
Even if we suppose that remains of Ru-compounds were physisorbed to the bacteria, the bacteria, when lysed, must have been broken into fragments that are small enough to pass through the filters we used (0.22 μm Millipore syringe filters), and this should have been detected.As such, in our opinion, we don't think we should modify the washing procedure.
6.The purity of commercial antibiotics should be included.
 The purity of the antibiotics has been added.

Reviewer #2:
This manuscript reports on the preparation of a series of diruthenium organometallic complexes employing a variety of functionalized ligands including BODIPY fluorescent probes and some lactam containing rings.Overall the synthesis of the organometallics is carried out according to synthetic procedures that are standard in this area and the characterization data appears to be consistent with the proposed structures.The rationale for this entire work is not fully clear -other than in vitro testing of some organometallics.It is far from clear if these molecules would have any utility as a clinical therapeutic.Glaring in the absence is the inclusion of any discussion of the potential human toxicity of ruthenium complexes and the barrier that might generate to any further utility of these molecules.A major scientific shortcoming of the work reported in the manuscript is any preliminary testing on mammalian cell lines.At a minimum a test in a cell line such as HEK293 would potentially provide some insight into the utility of these organometallic complexes.The time and expenses of mammalian cell line testing is acknowledged but in the case of these organometallics it would be necessary to ensure that this work is viewed with the appropriate impact.A conclusive demonstration of selectivity for bacterial cells over mammalian would certainly provide some helpful rationale for this project.
At present this work offers little of significant impact and would not generate much interest in the medicinal chemistry community.
 We thank this referee for his critical but fair assessment of the submitted version.We agree that providing existing data on the toxicity against Human Foreskin Fibroblasts (HFFs) of these compounds (published in Eur J Med Chem 2021, 222:113610, ChemBioChem, 23, e202200536, 2022, Molecules, 28, 902, 2023, Journal of Organometallic Chemistry 2023, 986:122624. In summary: Compounds 3, 5, 6, 12, 13, 15, 16 were ranked as very promising, with no toxic effect on HFF cells at a high concentration of 2.5 μM, compounds 1, 2, 7, 8 were moderately toxic, and compounds 9 and 10 were very toxic on HFF.We have compiled the data in a table included in the supplementary material (table S1) and added a paragraph in the main text mentioning toxicity and selectivity. Evaluation of the toxicity of compounds 17-22 against HEK293 cells has been performed (manuscripts still under preparation).The IC50 values range between 0.18 and 7.2 μM, and the selectivity Index (IC50 HEK293 / IC50 A24) between 1.2 and 3.

Specific comments:
Complex stability: the authors carry out a stability study by leaving samples in DMSO-d6 -however it is not clear if this was left in an NMR tube and how it was stored for the intervening 100 days.This is also not a very relevant stability test and perhaps a test with at least some concentration of water (not D2 O) present in the DMSO would be more biologically relevant.
 The samples dissolved in DMSO were left in the NMR tube and stored in the laboratory at room temperature, or at 4°C in the dark (BODIPY-compounds).
Concerning the presence of a small amount of H2O, the 1 H-NMR spectra of BODIPY compounds provided in ChemBioChem, 23, e202200536, 2022 (compounds 13-16) or in Journal of Organometallic Chemistry 2023, 986:122624 (compounds 9-10) show that there is already a non negligible amount of water (H2O) in the NMR tube.
NMR Characterization: Data: In the supporting information the authors report the 13C-NMR chemical shifts to 2 decimal places whereas one decimal place it more appropriate even at 100 MHz.Unless the authors have made changes to the default Bruker settings for collecting a broad-band decoupled spectrum then these should be indicated.
We thank this referee for pointing out this point.Our 13 C-NMR spectra are recorded with 64k points (standard acquisition parameters, acquisition time of 1.2s), leading to a resolution (Hz per point) of 0.5 Hz, thus 0.005 ppm. it is therefore possible to give an accuracy of 0.01 ppm.For example, for compound 17, in the13 C spectrum, the two carbons at 143.97 and 143.92ppm are perfectly discernible.As many of the reported carbon provide NMR resonances very close to each other, we prefer to keep the chemical shifts with a precision of 2 decimlas. julien.furrer@dcb.unibe.chhttp://furrer.dcb.unibe.ch