Synthesis, Reactivity Studies, and Cytotoxicity of Two trans-Iodidoplatinum(II) Complexes. Does Photoactivation Work?
Round 1
Reviewer 1 Report
The preparation, reactions with GMP with and without radiation, and cytotoxicity of two new trans-di-iodoplatinum(II) complexes are reported. The work is of a high standard and the results will be of interest to the readers of Inorganics. However, the manuscript needs some attention prior to acceptance as detailed below.
1. The grammar is poor and therefore extensive editing is required.
2. Lines 158-159: it is stated that Pt-N-C angles are opened up, but it is not stated which such angles. Also, it is suggested that the angles are “nearer to the tetrahedral geometry than expected”, but the expected angles are tetrahedral, so it is not clear what is meant here.
3. Microanalysis results have not been reported. If they are not available, evidence of purity is required such as high-quality NMR spectra.
Author Response
We would like to thank the positive evaluation of the referee 1. Below our answers point by point:
The preparation, reactions with GMP with and without radiation, and cytotoxicity of two new trans-di-iodoplatinum(II) complexes are reported. The work is of a high standard and the results will be of interest to the readers of Inorganics. However, the manuscript needs some attention prior to acceptance as detailed below.
1. The grammar is poor and therefore extensive editing is required.
We have tried to do our best with the editing of the manuscript. We have detected many mistakes and accordingly we have changed: some sentence structures, kept the the tenses in the paragraphs, corrected plurals, add proper linking words and shorten phrases.
2. Lines 158-159: it is stated that Pt-N-C angles are opened up, but it is not stated which such angles. Also, it is suggested that the angles are “nearer to the tetrahedral geometry than expected”, but the expected angles are tetrahedral, so it is not clear what is meant here.
We apologize for the poor quality of this paragraph, the concept is very simple: the molecule structure’ angles and distances show a slight deviation from the theoretical values. We have mended this paragraph and highlighted in yellow.
3. Microanalysis results have not been reported. If they are not available, evidence of purity is required such as high-quality NMR spectra.
We apologize for this negligence. We performed microanalysis as a mandatory assay for our procedure. Accordingly, we have added the values.
Reviewer 2 Report
In this manuscript, two trans diodido Pt(II) complexes with mixed nitrogen donor ligands are synthesized, characterized, tested in cancer cells, and evaluated for reactivity and photoreactivity with model nucleobases and imidazole. The key interesting result from this manuscript is that the pyridine ligand can be lost upon photolysis of the guanine adducts. This result has important implications on the further development of Pt iodide complexes as photoactivated drug candidates. Publication in Inorganics is recommended. However, there were a few issues with the manuscript in its current form that need to be revised prior to its unmitigated acceptance for publication. These concerns are listed below on a point by point basis:
1) In the Results section under Synthesis and characterization, it would still be useful to the readers to briefly describe the synthetic route used to obtain complex 1 and 2.
2) For Figure 2, the authors should use thermal ellipsoid diagrams if possible to more accurately represent the X-ray crystallographic data.
3) In Table 2, it should be specified that the numbers shown are IC50 values and that the numbers in the parentheses are the standard deviations. Otherwise the reader is left to assume this to be the case. Additionally, there are too many significant figures used for the IC50 values given the size of the associated standard deviations. Also, the large error for the complex 1 in SAOS-2 is noteworthy. Were additional replicates attempted to minimize this error?
4) For the NMR studies described in the Results section, the solvent is not specified. This information is only found in the experimental. It would be useful to specify the solvent both in the text and in the Figure captions.
5) The NMR data are a bit unclear. It would be helpful if the authors attempted to label all peaks within the spectra to assign them to specific complex. This would apply to Figure 3, 4, and 5. Why is the new peak at 8.2 ppm in Figure 3 a doublet? That should be explained as well.
6) The results of the DFT calculations do not appear to be as clear cut as the authors make them out to be. There is a lot going on with the EDDMs in Figure 6 and the assertion that they are dissociative to the pyridine ligand seems unfounded… It is clear that these transition represent a dxz (pi-non bonding) to dx2-y2 (sigma antibonding). The sigma antibonding nature of this accepting orbital will be antibonding with respect to all four of the ligands though. Why do the authors interpret this to show more antibonding character towards the pyridine? It seems like additional analysis, such as quantifying orbital contributions, would be necessary to make this claim. Alternatively, the authors could try optimizing the lowest energy triplet states as it is not unreasonable that the photochemistry of these heavy atom-containing molecules proceeds through the triplet state.
7) Given the emphasis of photoactivity, why wasn’t the phototoxicity evaluated?
8) As an additional control, did the authors investigate the irradiation of complexes 1 and 2 in the absence of any donor ligands (guanine or MeIm)?
9) Figure 7 is a bit unclear. The loss of pyridine occurs only upon light irradiation. Somewhere in the figure it should be indicated that light is needed to trigger that final step.
10) What is the method of verifying the purity of the Pt(II) complexes? Only 1H NMR is provided as characterization in the experimental. NMR provides identity but not purity. Was elemental analysis or HPLC carried out for these compounds?
11) In the experimental, it is stated that the iodide complexes were dissolved in PBS to make 10 mM solution. That high level of solubility for these iodide complexes seems surprising. Can the authors confirm that no co-solvent was added? Additionally, was there any concern about substitution of iodide for chloride in the high chloride concentration solution of PBS?
Author Response
In this manuscript, two trans diodido Pt(II) complexes with mixed nitrogen donor ligands are synthesized, characterized, tested in cancer cells, and evaluated for reactivity and photoreactivity with model nucleobases and imidazole. The key interesting result from this manuscript is that the pyridine ligand can be lost upon photolysis of the guanine adducts. This result has important implications on the further development of Pt iodide complexes as photoactivated drug candidates. Publication in Inorganics is recommended. However, there were a few issues with the manuscript in its current form that need to be revised prior to its unmitigated acceptance for publication. These concerns are listed below on a point by point basis:
1) In the Results section under Synthesis and characterization, it would still be useful to the readers to briefly describe the synthetic route used to obtain complex 1 and 2.
We have briefly described the method with special attention to the slight variations performed.
2) For Figure 2, the authors should use thermal ellipsoid diagrams if possible to more accurately represent the X-ray crystallographic data.
We have replaced the balls and stick diagram of figure 2 by Ellipsoid diagrams.
3) In Table 2, it should be specified that the numbers shown are IC50 values and that the numbers in the parentheses are the standard deviations. Otherwise the reader is left to assume this to be the case. Additionally, there are too many significant figures used for the IC50 values given the size of the associated standard deviations. Also, the large error for the complex 1 in SAOS-2 is noteworthy. Were additional replicates attempted to minimize this error?
We agree with the reviewer´s opinion, we must be cautious with this experimental value. Following the recommendations, we have specified the values and added a note in table 2 to clarify the impact of this SD for the complex 1 in SAOS-2. We performed the assay by triplicate unfortunately; we could not get a better SD value.
4) For the NMR studies described in the Results section, the solvent is not specified. This information is only found in the experimental. It would be useful to specify the solvent both in the text and in the Figure captions.
In revised version, we have specified the solvent used in the experiments within discussion and in the figure captions.
5) The NMR data are a bit unclear. It would be helpful if the authors attempted to label all peaks within the spectra to assign them to specific complex. This would apply to Figure 3, 4, and 5. Why is the new peak at 8.2 ppm in Figure 3 a doublet? That should be explained as well.
We apologize for the limited labelling within the spectra. We have labelled the peaks in the spectra of the figures 3, 4 and 5.
Regarding the peak at 8.2 ppm in Figure 3, we think this signal could be assigned to one of the pyridine protons of a new specie from the speciation discussed in the article. For example the 1-py* adduct could afford aqua species, but at this stage is not wise to assure this assignation. We have tried to perform other NMR experiments, 2D NMR requires long accumulation experiments and the sample evolves, so the results are inconclusive. The titration of the sample produces a milky solution, which highly hampered the measure.
We have included a new figure (Figure S1 new version) in the SM with the free-py and the free 5-GMP spectra together with the integrals of two selected spectra: at 3 h and initial. In spite of being a mixture, the signals at the initial spectra (and at 3h) are very indicative. Which is clear to us, is that this doublet is not free-pyridine.
6) The results of the DFT calculations do not appear to be as clear cut as the authors make them out to be. There is a lot going on with the EDDMs in Figure 6 and the assertion that they are dissociative to the pyridine ligand seems unfounded… It is clear that these transition represent a dxz (pi-non bonding) to dx2-y2 (sigma antibonding). The sigma antibonding nature of this accepting orbital will be antibonding with respect to all four of the ligands though. Why do the authors interpret this to show more antibonding character towards the pyridine? It seems like additional analysis, such as quantifying orbital contributions, would be necessary to make this claim. Alternatively, the authors could try optimizing the lowest energy triplet states as it is not unreasonable that the photochemistry of these heavy atom-containing molecules proceeds through the triplet state.
We added as example the LUMO orbitals of the mono and bis 9-EtG adducts participating to the lowest-energy transitions. The reviewer is right, they all have antibonding character towards the four ligands (see Supporting Information). However, if one looks at the calculated transition for 1 and 2 (and hence at the LUMO orbitals contributing to them), they barely involve the pyridine ligands. These aspects suggest that irradiation of the 9-EtG adducts is more likely to result in pyridine release. Surely, this is qualitative explanation and more advanced studies (such as looking at the triplets and their potential energy surfaces) would provide more insights. This type of work is however out of the scope of this manuscript and would require a significant amount of extra experimental work. We have improved the explanation in the text.
7) Given the emphasis of photoactivity, why wasn’t the phototoxicity evaluated?
This is a very interesting point, and we thought about it, but then we observed the release of the spectator ligand, we concentrate in analyzing their behavior in order to be able to understand what species could act as the active ones. As we propose, the photoactivation will afford species that we have already studied and published, so we believe that, in some way, have shown the value of this procedure.
Moreover, we think that the importance of our work was to find out that for the first time for trans platinum complexes.
8) As an additional control, did the authors investigate the irradiation of complexes 1 and 2 in the absence of any donor ligands (guanine or MeIm)?
Yes we did. We studied the stability of the complexes at dark up to 24 h, observing no release. Once we irradiate, the complexes were stable up to 6h as well.
9) Figure 7 is a bit unclear. The loss of pyridine occurs only upon light irradiation. Somewhere in the figure it should be indicated that light is needed to trigger that final step.
Figure 7 has been modified accordingly and we think now is clearer
10) What is the method of verifying the purity of the Pt(II) complexes? Only 1H NMR is provided as characterization in the experimental. NMR provides identity but not purity. Was elemental analysis or HPLC carried out for these compounds?
As mentioned to the previous referee, this is a negligence in our part. We apologize again for this mistake. We performed microanalysis, the data are included now.
11) In the experimental, it is stated that the iodide complexes were dissolved in PBS to make 10 mM solution. That high level of solubility for these iodide complexes seems surprising. Can the authors confirm that no co-solvent was added? Additionally, was there any concern about substitution of iodide for chloride in the high chloride concentration solution of PBS?
We really appreciate this comment because we have realized that this was a mistake in the description of the sample preparation and therefore we have corrected it.
Reviewer 3 Report
The manuscript describes the synthesis and characterization of two trans-diiodido-platunum(II) complexes. Moreover, the cytotoxicity of the prepared complexes against five human cancer cell lines has been evaluated together with the solution behaviour of the compounds, and influence of photoactivation on some biological features has been studied. The DFT calculations have been used to describe some spectral and bonding properties within the vicinity of the central platinum atom. In general, I consider the work to be useful trying to solve an actual topic in the field of bioinorganic chemistry. However, on the other side, reading the text, some comments, questions and suggestions arose and they are as follows:
Line 2, Title: I would suggest the authors to modify the title as follows: insert the term “trans” into … cytotoxicity of two trans-diiodido-platinum(II) complexes.
Line 85, Table 2: Time of incubation should be added into the legend. The standard deviation in the case of complex 1 related to the SAOS-2 cell line is too high, probably, this value is influenced by a gross error during the experiment and this should be excluded or the corresponding experiments should be repeated.
Line 106, Figure 3: I suggest the authors to add 1H NMR spectra of free 5-GMP and free pyridine to reveal if the pyridine remains coordinated to platinum or it cleaves off after adding of 5-GMP.
Line 203: The purity of the samples should be supported by suitable techniques. I suggest to determine the contents of C,H and N using elemental analysis and to show 1H NMR spectra to reveal the purity.
ESI, Table S2: The Pt–I bond lengths between the calculated and X-ray diffraction determined differ significantly. I mean that a more proper basis set for non-platinum atoms should be used (e.g. tzvp) in the case of the calculations. This comment is also valid for the whole chapter 2.4. DFT calculations.
Line 264: I would rephrase Conclusions as follows:
1/ instead of the term “cytotoxic activity” I would suggest use the term “cytotoxicity”. (given also in lines 43, 51)
2/ I mean that the following sentence is not fully supported by experimental data “… this result provides evidences that the aromatic nature of the amine ligand plays an important role in the photochemistry of trans diiodido platinum complexes and also that they can be considered as potential photoactivable prodrug compounds following two different pathways.“, because just two complexes have been studied. The group of the compounds under investigations should be extended to support the above-given statement.
In my opinion, the main lack of the work lies in the fact that no cytotoxicity of the complexes 1 and 2 was determined after irradiation of the samples because this could reveal that the photoactivated complexes 1 and 2 could show higher cytotoxicity than cisplatin. Moreover, no correlation between the cytotoxicity and increased reactivity towards model molecules has been studied after irradiation. In addition, the toxicity against healthy cells would also show the selectivity of the studied platinum complexes 1 and 2.
I believe that after acceptance of the above-said suggestions the manuscript could be accepted for publication in Inorganics.
Author Response
The manuscript describes the synthesis and characterization of two trans-diiodido-platunum(II) complexes. Moreover, the cytotoxicity of the prepared complexes against five human cancer cell lines has been evaluated together with the solution behaviour of the compounds, and influence of photoactivation on some biological features has been studied. The DFT calculations have been used to describe some spectral and bonding properties within the vicinity of the central platinum atom. In general, I consider the work to be useful trying to solve an actual topic in the field of bioinorganic chemistry. However, on the other side, reading the text, some comments, questions and suggestions arose and they are as follows:
Line 2, Title: I would suggest the authors to modify the title as follows: insert the term “trans” into … cytotoxicity of two trans-diiodido-platinum(II) complexes.
We have inserted the term trans in the title in agreement with the referee’s suggestion
Line 85, Table 2: Time of incubation should be added into the legend. The standard deviation in the case of complex 1 related to the SAOS-2 cell line is too high, probably, this value is influenced by a gross error during the experiment and this should be excluded or the corresponding experiments should be repeated.
Right!. This value is not our best result. We have highlighted the problem as recommended by referee 1, but as an experimental value, we think is valuable. As mentioned in our previous comments, we have repeated the values with no better results. Therefore, if the referee require so, we will delete the value from the table.
Line 106, Figure 3: I suggest the authors to add 1H NMR spectra of free 5-GMP and free pyridine to reveal if the pyridine remains coordinated to platinum or it cleaves off after adding of 5-GMP.
In revised version, we have included new spectra (Figure s1) that includes the 5-GMP and pyridine spectra using the same experimental conditions and pilled up with two of the spectra of Figure 3. Pyridine remains coordinated.
Line 203: The purity of the samples should be supported by suitable techniques. I suggest to determine the contents of C, H and N using elemental analysis and to show 1H NMR spectra to reveal the purity.
ESI, Table S2: The Pt–I bond lengths between the calculated and X-ray diffraction determined differ significantly. I mean that a more proper basis set for non-platinum atoms should be used (e.g. tzvp) in the case of the calculations. This comment is also valid for the whole chapter 2.4. DFT calculations.
The choice of the functional and basis set was made after benchmarking work was performed on complex 2. We (erroneously) did not introduce it in the manuscript for sake of brevity. The benchmarking data is here reported and now added to the supporting information of the manuscript. The reviewer might be right that the tzvp basis set for non-platinum atoms is likely to give better results. Nonetheless, we believe our benchmarking work is already significant (well above the standard of manuscripts in this field) and the discrepancy in the Pt–I bond distances we have obtained are acceptable within the DFT approximation. We have to consider that we are ultimately comparing an experimental solid state structure with a theoretical one calculated in solution for an isolated molecule.
Benchmarking calculations:
Table S2b. Selected bond distances (Å) for complex 2 optimized with the DFT method at the PBE1PBE:ECP/6-31G** level using different ECPs for the Pt atom and the PCM solvent (water) model.
6-31G** PCM (water) | |||||||
Complex 2 | X-ray | LANL2DZ | D | LANL08 | D | SDD | D |
Pt–I(2) | 2.596 | 2.706 | 0.110 | 2.687 | 0.091 | 2.692 | 0.096 |
Pt–I(3) | 2.592 | 2.706 | 0.114 | 2.687 | 0.095 | 2.692 | 0.100 |
Pt–N(8) | 2.067 | 2.068 | 0.001 | 2.065 | -0.002 | 2.070 | 0.003 |
Pt–N(20) | 2.025 | 2.032 | 0.007 | 2.030 | 0.005 | 2.037 | 0.012 |
Table S2c. Selected bond distances (Å) for complex 2 optimized with the DFT method at the PBE1PBE:LANL08/BS level using different basis sets (BS) for the non-Pt atoms and the PCM solvent (water) model.
LANL08 PCM (water) | ||||||||
Complex 2 | X-ray | 6-31G** | D | 6-31+G** | D | 6-311G** | D | |
Pt–I(2) | 2.596 | 2.687 | 0.091 | 2.683 | 0.087 | 2.682 | 0.086 | |
Pt–I(3) | 2.592 | 2.687 | 0.095 | 2.683 | 0.091 | 2.682 | 0.090 | |
Pt–N(8) | 2.067 | 2.065 | -0.002 | 2.064 | -0.003 | 2.065 | -0.002 | |
Pt–N(20) | 2.025 | 2.030 | 0.005 | 2.030 | 0.005 | 2.032 | 0.007 | |
6-311+G** | D | |||||||
2.685 | 0.089 | |||||||
2.685 | 0.093 | |||||||
2.064 | -0.003 | |||||||
2.032 | 0.007 | |||||||
Table S2d. Selected bond distances (Å) for complex 2 optimized with the DFT method using different functionals, the LANL08/6-31G** ECP/basis set and the PCM solvent (water) model.
LANL08/6-31G** PCM (water) | |||||||||
Complex 2 | X-ray | PBE1PBE | D | LC-wPBE | D | ||||
Pt–I(2) | 2.596 | 2.687 | 0.091 | 2.664 | 0.068 | ||||
Pt–I(3) | 2.592 | 2.687 | 0.095 | 2.664 | 0.072 | ||||
Pt–N(8) | 2.067 | 2.065 | -0.002 | 2.059 | -0.008 | ||||
Pt–N(20) | 2.025 | 2.030 | 0.005 | 2.032 | 0.007 | ||||
B3LYP | D | CAM-B3LYP | D | M062X | D | ||||
2.739 | 0.143 | 2.707 | 0.111 | 2.707 | 0.111 | ||||
2.739 | 0.147 | 2.707 | 0.115 | 2.707 | 0.115 | ||||
2.090 | 0.023 | 2.074 | 0.007 | 2.072 | 0.005 | ||||
2.056 | 0.031 | 2.046 | 0.021 | 2.046 | 0.021 | ||||
Line 264: I would rephrase Conclusions as follows:
1/ instead of the term “cytotoxic activity” I would suggest use the term “cytotoxicity”. (given also in lines 43, 51)
According to the referee’s suggestion, we have replaced the term cytotoxic activity by cytotoxicity along the manuscript
2/ I mean that the following sentence is not fully supported by experimental data “… this result provides evidences that the aromatic nature of the amine ligand plays an important role in the photochemistry of trans diiodido platinum complexes and also that they can be considered as potential photoactivable prodrug compounds following two different pathways.“, because just two complexes have been studied. The group of the compounds under investigations should be extended to support the above-given statement.
We agree with the referee and therefore we have modified this conclusion
In my opinion, the main lack of the work lies in the fact that no cytotoxicity of the complexes 1 and 2 was determined after irradiation of the samples because this could reveal that the photoactivated complexes 1 and 2 could show higher cytotoxicity than cisplatin. Moreover, no correlation between the cytotoxicity and increased reactivity towards model molecules has been studied after irradiation. In addition, the toxicity against healthy cells would also show the selectivity of the studied platinum complexes 1 and 2.
As we mentioned previously, this a very interesting point and we thought about it, but then we observed the release of the pyridine and tried to find out the active species. As we propose, the photoactivation will afford species that we have already studied and published, so we, in some ways have the value of this procedure.
I believe that after acceptance of the above-said suggestions the manuscript could be accepted for publication in Inorganics.
Thanks for the helpful comments that we believe have increased the quality of the paper.
Round 2
Reviewer 3 Report
I can state that the authors performed the appropriate changes which led to the improvement of the manuscript and thus, I suggest its acceptance. There is just one minor issue for solving. I would suggest to add the formulas of the complexes into Abstract to help readers to see what type of complexes is presented and studied.
Author Response
thank you for the acceptance of the manuscript. We have included the requiered formulae.
